:i~RI~~rjmorris/sciev.06/SIMULATIONS/stanlaw_5... · 1 5,186,827 2 says, involve the use of labeled antibodies instead of labelled analyte. In performing an immunometrk assay,

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PAUL A . L I B E R T I , C H U R L H V I L L E , PA; B R I A N P . FEELEY, EASTON,.PA; DHANESH I . GOHEL, P H I L A D E L P H I A , PA.

. FORE I'GN F'I L 1 NG L I CENSE GRANTED 04/06/91

DANN, DORFMAN, HERRELL AND S K I L L M A N 1310 THE FIDELITY BUILDING

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$ 123 S. BROAD STREET P H I L A D E L P H I A , PA 19109

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f: I- MAGNETIC MEANS

APPARATUS AND METHODS F O R MAGNETIC SEPARATION FEATURING EXTERNAL

This i s t o c e r t i f y that annexed hereto i s a t rue copy from the records of the United States Patent and Trademark O f f i c e of the appl lcat lon as o r i g i n a l l y filed whlch i s i d e n t i f i e d above.

By authori ty of the COMMISSIONER OF PATENTS AND TRADEMARKS

Date Cert i fv ina .officer

111111 llllllll Illllf ll lllll1111111111111111111111111 lllll111111111111111111 _ _ US005186827A

United States Patent, [I91 1111 Patent Number: 5,186,827 Liberti et al. [45] Date of Patent: Feb. 16, 1993

I541 APPARATUS ,FOR MAGNETIC SEPARATION FEATURING EXTERNAL MAGNETIC MEANS

I751 Inventors: Paul A. Liberti, Churchville; Brian P. Feeley, Easton; Dbnnesh I. Gobel, Philadelphia, all of Fa.

Valley, Pa. [73] Assignee: Immuaieon Corporation, Huntington

1211 Appl. No.: 674,678

[22] Filed: Mar. 25, 1991

I5 11 Int. C l . 5 .............................................. BOlD 35/06 .- - A

[52] US. Cl. .................................... 210/22& 210/223; 422/101

[58] Field of Search ....................... 210/222, 695, 223; 209/224, 228, 232; 435/2; 436/177, 526;

422/101; 335/306 ~561 References Cited

U.S. PATENT DOCUMENTS 564,858 7/1896 Whitacre et al. ................... 209/232

3,402,820 9/1968 Lohmann ............................ 210/222 3,567,026 3/1971 Kolm ................................... 210/222 3,676.331 7/1972 . Kolm ................................... 210/222 3,902,994 9/1975 Maxwell et al. .................... 209/214 3,970,518 7/1976 Giaever ................................ 19V1.5 3,985,649 10/1976 Eddelrnan ........................... 210/695

4,261,234 5/1961 ........................... 4,452,713 6/1984 4,498,907 2/1985 4,554,088 11/1985 4,659,678 4/1987 4,663,029 5/1987 4,795,698 1/1989 ..................... 4,895,650 1/1990 Wang .................................. 210/222

1 OTHER PUBLICATIONS Zmmunwssaysfir Clinical Chemistry, pp. 147-162, Hun- ter et al. editors. The Properties of Mugnetic Supports in Relution to Immo-

bilized Enzyme Reactors, Robinson et al., Biotechnology and Bioengineering, vol. XV (1973). The Dynal MPC-1 (manufactured by Dynal, inc., Great Neck, N.Y.)-product information sheet (1987). BioMag Separator (manufactured by Advanced Mag- netics, Inc., Cambridge, Mass.)-catalog pages (3 sheets). Magnetic separator (manufactured by Ciba-Corning Medical Diagnostics, Wampole, Mass.>catalog cover and catalog pages (2 sheets). Magnetic Separator System (manufactured by Serono Diagnostics, Nonvell, Mass.)-catalog pages (2 sheets). Magnetic separator (made by Miltery Biotech GmbH, Gladbach, Germany)-product information literature. High Gradient Magnetic Separation Theory and Appli- cations, R. R. Oder, IEEE Transactions on Magnetics, vol. MAG-12, No. 5, Sep. 1976. Magnetite-Protein Conjugates for the Separation of Cells by High Gradient Magnetic Filtration, C. S. Owen et al., Cell Separation Methods and Selected Applications, vol. 4, 1987. Magnetic Separation Techniques- Their Application to Medicine, J. T. Kernshead et al., Molecular and Cellular Biochemistry 67. 11-18 (1985). Magnetic Solid-Phase Radioimmunoassy, L. S. Hersh et al., CIinica Chimica Acta, 63 (1975) 69-72.

Primary Examiner-Robert A, Dawson Assistunt Examiner-Matthew 0. Savage Attorney, Agent, or Firm-Dann, Dorfmann, Herrell and Skillman WI ABSTRACT A magnetic separator for separating magnetic particles from a non-magnetic test medium is disclosed herein. The magnetic separator includes a non-magnetic container having a peripheral wall with an internal surface area for receiving the test medium, and magnetic means for generating a magnetic field gradient within the container in which the magetic field is stronger in the test medium along the internal surface area of the peripheral wall than in the medium most distant from the wall.

38 Claims, 2 Drawing Sheets

U.S. Patent Feb. 16, 1993 Sheet 1 of 2 5,186,827

FIG. 1

I

FlG.2 FIG. 3

U.S. Patent Feb. 16, 1993 Sheet 2 of 2 5,186,827

6

FIG. 5

FI G.

363 tZ5 f 323 ( 1

361

fig.6

1 5,186,827

2 says, involve the use of labeled antibodies instead of labelled analyte. In performing an immunometrk assay, a sandwich is formed in which the "layers" are: antibody/multivalent (minimally bivalent) antigedanti-

The amount of the labeled antibody which is b u n d for each complete sandwich complex (antibody/an-

. tigedantibody) is directly proportional to the amount of target antigenic substance present in the test sample.

lo Sandwich assays can be performed in multi-step fashion with polyclonal antibodies or in fewer steps when

5 body.

APPARATUS FOR MAGNETIC SEPARATION FEATURING EXTERNAL MAGNETIC MEANS

mon&lonals directed to independent antigenic determi-

FIELD OF THE INVENTION The present invention is directed to magnetic separa-

tion apparatus and methods in which magnetic particles are used for isolating substances of interest from a non- magnetic test medium by means of high gradient magnetic separation (HGMS).

BACKGROUND OF THE INVENTION The present invention relates to improvements in

magnetic separators and methods of separation Of mag- In both the conventional competitive immunoassay netic particles from non-magnetic media, having partic- ' 5 and the Wunometric assay just described, quantifica- Ular Utility in Various laboratory and clinical procedures tion of the target substance requires a *para. involving biospecific affinity reactions. Such reactions tion of bound from fret labeled ligand or labeled recep- are commonly employed in testing biological samples, tor. such as blood or urine, for the determination of a wide Bound/free separations may be accomplished gravi-

such as cells, proteins, nucleic acid sequences, and the gation of finely divided particles or beads coupled to the target substance. If desired, such particles or beads like.

any member of a specific binding pair, i.e., a pair of ration step. Magnetic particles are well known in the substances or a substance and a structure exhibiting a 25 mutual affinity of interaction and includes such things as art* as is their use in immune and Other bio-spcific

gmd" is used herein to refer to substances, such as 4,554,088 and Immunoassays for c h i c a l Chemistry. pp. antigens, haptens and various cell-associated StmCtUreS, 147-162, Hunter et al. eds., Churchill Livingston, Edin- having at Ieast one characteristic determinant or epj- 30 borough (1983). GeneralIy, any material which facill- tope, which are capable of being biospecifically recog- tates magnetic Or gravitational =Paration may be em- nized by and bound to a receptor. "Receptor" is used ployed for this purpose. herein to refer to any substance or group of substances Small magnetic particles have proved to be quite having a biospecific binding affinity for a given ligand, useful in analyses involving biospecific affinity reac- to the substantial exclusion of other substances. Among 35 tions, as they are conveniently coated with biofunc- the receptors determinable via biospecific affinity reac- tional polymers, e.g., proteins, provide very high sur- tions are antibodies (both polyclonal and monoclonal), face areas and give reasonable reaction kinetics. Mag- antibody fragments, enzymes, nucleic acids, Clq and the netic particles ranging from 0.7-1.5 microns have been like. The determination of any member of a biospecific described in the patent literature, including, by way of binding pair is dependent upon its selective interaction 40 example, U.S. Pat. Nos. 3,970,518; 4,018,886; 4,230,685; with the other member of the pair. 4,267,234; 4,452,773; 4,554,088; and 4,659,678. Certain

Various methods are available for determining the of these particles are disclosed to be useful solid SUP- above-mentioned target substances based upon complex ports for immunologic reagents, having reasonably formation between the substance of interest and its spe- good suspension charactefistics when mildly agitated. cific binding partner. Means are provided in each in- 45 Insofar as is known, however, absent degree of stance whereby the occurrence Or degree Of target agitation, all ofthe magnetic particles presently in com- substancehinding partner complex formation is deter- mercial use settle in time and must be resuspended pior

to use. This adds another step to any process employing minable. In the case of a competitive immunoassay to deter- such reagents. mine antigen, for example, the antigen of interest in a 50 Small magnetic particles, such Bs those mentioned

test -'le competes with a known quantity Of labelled above, generally fall into two broad categories. The antigen for a limited quantity of specific antibody bind- category includes particles that Bre pemancntly amount of labelled antigen bound to specific antibody is inversely proportional to the quantity of antigen in the 55 become magnetic Only when subjected to a

nants are employed.

range Of target substances, specially biological entities 2o tationally, e.g. by senrig, or, alternatively, by centfifu-

As used the term "target Substance" refers to may be made to facilitate the bound/free sepa-

cell components, biospecific ligands and receptors. "Lis affinity reactions. See, for u.s* Pat.

'g sites' after appropriate reaction period the magnetized; and the second comprises that

sample. ampe t i t i ve assays for antibodies, field. The latter are referred to herein as magnetically

rather than labeled antigen, function in an analogous responsive behavior are sometimes dcmibed as super- mamer. resulting immune complexes are =pa- paramgnetic. However, certain ferromagnetic materi- rated, for example, by immunoabsorption, physico- 60 als, magnetic iron oxide, may be characterized as chemical adsorption or precipitation of either the corn- ma@eticallY resPOnSiVe when the size is about plexes or unbound antigen, Antibodybound labeled 300A Or less in diameter. Larger CrYStdS Of ferromag- antigen is then quantified and a standard curve is con- netic materials, by Contrast, retain permanent magnet strutted from hewn antigen concentrations, from characteristics after exposure to a magnetic field and which unknown concentrations of antigen may be de- 65 tend to aggregate thereafter. See P. Robinson et d., termined. Biotech Bioeng. XV:603-06 (1973).

In contrast, immunometric assays for the determina- Magnetically responsive colloidal magnetite is tion of antigen, commonly known as "sandwich" as- known. See U.S. Pat. No. 4,795,698 to Owen et a].,

ing la&led antibodies (typically monoclonal antibodies) responsive Particles. Materials displaying mapeticdly

5.186,827 3

which relates to polymer-coated, sub-micron size magnetite particles that behave as true colloids.

The magnetic separation apparatus/method used for bound-free separations of target substance-bearing magnetic particles from test media will depend on the nature and particle size of the magnetic particle. Micron size ferromagnetic, i.e., permanently magnetized, particles are readily removed from solution by means of commercially available magnetic separation devices. These devices employ a single relatively inexpensive permanent magnet located external to a container holding the test medium. Examples of such magnetic separators are the MAIA Magnetic Separator manufactured by Serono Diagnostics, Nonvell, Mass., the DYNAL MPC-1 manufactured by DYNAL, Inc., Great Neck, N.Y. and the BioMag Separator, manufactured by Ad- vanced Magnetics, Inc., Cambridge, Mass. A specific application of a device of this type in performing magnetic solid-phase radioimmunoassay is described in L. Hersh et al., magnetic separator, manufactured by Ciba- Corning Medical Diagnostics, Wampole, Mass. is provided with rows of bar magnets arranged in parallel and located at the base of the separator. This device accommodates 60 test tubes, with the closed end of each tube fitting into a recess between two of the bar magnets.

An automated continuous-flow radioimmunoassay system employing cellulose-coated magnetic particles is described in U.S. Pat. No. 4,141,687. The automated system exemplified in the '687 patent includes elaborate electromagnetic traps which are operable in a pre-determined sequence by a programmer device under the control of a sample detector.

The above-described magnetic separators have the disadvantage that the magnetic particles attracted toward the magnets tend to form in multiple layers on the inner surface of the sample container where they are entrapped along with impurities that are difficult to remove even with vigorous washing.

Colloidal magnetic materials are not readily separable from solution as such, even with powerful electro-mag. nets but, instead, require high gradient field separation techniques. See, R. R. Oder, IEEE Trons. Magnetics, 12: 428-35 (1976); C. Owen and P . Liberti, Cell Separation: Methods and Selected Applications, Vol. 5, Pretlow and Pretlow eds., Academic Press, N.Y., (1986); J. T. Kems-

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45 . . head and J. Ugelstad, Magnetic Molecular ond Cellular Biochern., 67,ll-18 (1985). The gradient fields normally used to filter such materials generate hugh magnetic forces. Another useful technique for performing magnetic ,separations of colloidal magnetic particles from a 50 test medium, by various manipulations of such particles, c.g., addition of agglomerating agents, is the subject of co-pending and commonly owned U.S. patent application Ser. No. 389,697, filed Aug. 4, 1989.

cally accomplished by using a device having a separation chamber in which a wad of magnetic stainless steel wire is disposed between the poles of a conventional electro- or superconducting magnet and serves to generate large field gradients around the wire which exert 60 a strong attractive force on target substance-bearing magnetic particles.

A commercially available high gradient magnetic separator of the type described immediately above is the MACS device made by Miltenyi Biotec GmbH, Glad- 65 back, West .Germany, which employs a column filled with a non-rigid steel wool matrix in cooperation with a permanent magnet. In operation, the enhanced mag-

High gradient magnetic separation (HGMS) is typi- 55

4 netic field gradient produced in the vicinity of the steel wool matrix attracts and retains the magnetic particles while the non-magnetic test medium passes through and is removed from the column. Similar magnetic separators employing a steel wool matrix for separating colloidal size magnetic components from a slurry containing same are also disclosed in U.S. Fat. Nos. 3,567,026, 3,676,337 and 3,902,994. In the last mentioned patent, the separator is provided with a magnetic wool matrix capable of movement into and out of the influence of a magnetic field as a continuously moving element.

It has been found that the steel wool m a t h of such prior art HGMS devices often gives rise to non-specific entrapment of biological entities, other than the target substance, which cannot be removed completely without extensive washing and resuspension of the particles bearing the target substance. Moreover, the size of the column in many of the prior art HGMS devices requires substantial quantities of experimental materials, which limits their use in performing various important laboratory-scale separations. In addition, the steel wool matrix may be harmful to certain sensitive cell types.

A useful magnetic separator that avoids problems identified above is the subject of co-pending and commonly owned U.S. patent application Ser. No. 588,662, filed Sep. 26, 1990. The separator of this co-pending application comprises magnetic means featuring a pair of confronting magnets external to the container and a magnetic gradient intensifying means positioned within a container holding the test medium. The magnetic particles adhere to the magnetic means within the container which serves to separate or remove the particles from the test medium. U.S. Pat. No. 4,663,029 relates to an HOMS device

which is stated to be an improvement with respect to devices employing a magnetic wool matrix as the magnetic field gradient intensifier, as well as to devices relying on differences in magnetic susceptibility of particles in a fluid to effect separation. The '029 patent describes an apparatus for continuous magnetic separation of particles from a slurry according to their magnetic moment, by passing the slurry through a separator comprising a non-magnetic canister with a magnetized wire or rod extending adjacent to the canister. The wire is magnetized by a magnetic field to create a magnetization component transverse to the longitudinal axis of the wire, thereby to provide a field gradient extending everywhere within the canister space and exerting a radial force on particles passing through the canister. Depending upon the orientation of the magnetic field relative to the canister, diamagnetic particles in the slurry can be attracted toward the wire and paramagnetic particles repelled, or vice versa, for a magnetic field usually rotated by 90" with respect to the plane of the canister.

From the foregoing review of the prior art, it is apparent that HGMS affords certain advantages in performing medical or biological analyses based on biospecific afEnity reactions involving colloidal magnetic particles. Nevertheless, it would be desirable to provide HGMS apparatus and methods which are of relatively simple construction and operation, relying only on gradient intensifying means external to the separation chamber, and yet maximizing magnetic field gradients, and which reduce entrapment of nontarget substances, eliminate loss of immobilized target substance due to shear forces or collisions with other biological entities, and enable use of standard microtiter plate wells, and

5,186,827 5 6

the like. Such a development would clearly be of practi- ca1 utility in conducting various laboratory-scale separations, particularly in immunoassays and cell sorting.

and positioning of the magnets located on the opposite sides of the cavity are such as to produce flux lines which generate a high gradient magnetic field within - the testmedium in the container. The magnets may be

5 housed in a ferromagnetic yoke,.preferably of cylindri- SUMMARY OF THE INVENTION It is an object of this invention to provide magnetic cal configuration, which serves- to enhance the field

separation apparatus and methods capable of generating strength produced by the apparatus. The magnetic field a high gradient magnetic field within a non-magnetic gradient produced by this “multipole” arrangement is test medium to separate magnetically responsive colloi- characterized by a very strong magnetic field near the dal particles from the test medium. Unlike relatively 10 edge of the cavity and by virtually no magnetic field at larger size magnetic particles which tend to settle out of the center of the cavity. Accordingly, magnetic parti- an aqueous medium, magnetically responsive colloidal cles in the test medium adjacent the wall of the con- particles remain suspended in an aqueous medium for an tainer near the edge of the cavity are subject to consid- indefinite period, thereby making them readily accessi- erably greater magnetic force than particles in the test ble to target substances. 15 medium furthest from the wall of b e container, toward

The magnetic separator of the invention comprises at the center of the cavity where the field strength falls least one container and magnetic means capable of gen- away to zero. crating a high gradient magnetic field in the test me- The magnetic means may advantageously comprise dium within the container. The container has a pcriph- magnetic flux concentrating means, particularly, if the eral wall with an internal surface area and is adapted to 20 cavity defined by the magnets is much larger than the receive the test medium with the magnetically respon- cross-section of the container or carrier. Suitable for sive colloidal particles therein (hereinafter “the test this purpose are pole pieces of various geometries, medium being separated”). As will be described in fur- which are magnetized or magnetizabk via an induced ther detail below, the magnetic field gradient generat- field. The magnets comprising the magnetic means may ing means is disposed outside the container and pro- 25 be attached magnetically to, or otherwise joined to the vides an “open” field gradient inside the container, yoke, e.g. by cementing with epoxy, to hold the mag wherein the magnetic field is stronger in the test me- nets in a fued position relative to one another. dium along the internal wall surface of the container The physical properties of the magnetic particles than it is in the test medium most distant from the wall. preferably used in the practice of this invention, particu-

If the test medium being separated is in a steady state, 30 larly the relatively small particle size, permit a level of e.g., in a batch-type operation, suitable containers in- operating eficiency which, insofar as is known, has not clude microtiter wells, test tubes, capillary tubes closed been achievable heretofore. Furthermore, by control- at one end, or other nonmagnetic cylindrical walled ling the quantity of magnetic particles added to the test vessels defining a chamber for performing the desired medium, relative to the exposed surface area of the wall separation. Furthermore, a plurality of test samples may 35 of the container in contact with the test medium and be processed simultaneously through the use of a carrier controlling the orientation of such exposed surface, so capable of holding more than one sample container. In as to be substantially cocxtcnsive with the flux lines of a preferred form, the carrier includes means for holding the magnetic field, it is possible to cause the magnetic a plurality of containers around the periphery of the particles to adhere along the exposed surface of the carrier. 40 container wall in a substantially single layer, corre-

If the test medium is to pass continuously through the sponding in thickness to about the size of the magnetic separator, a suitable container is a conduit or tube hav- particles and any substance or material borne thereby. ing openings at each end. Such containers are prefera- By operating in this way, occlusion of nonspecifically bly non-magnetic, e.g., glass or plastic, and of cylindri- bound substances in the immobilized magnetic particles cal configuration. Preferably, the container has an inlet 45 is virtually negligible. opening at one end for receiving the test medium which In separating magnetically responsive colloidal parti- is exposed to the high magnetic field gradient at the cles from a non-magnetic test medium in accordance center portion of the container. In this particular em- with the methods of the invention, the particles are bodimenl, the container may also have one or more initially dispersed in the non-magnetic test medium, non-magnetic bames spaced apart within the container 50 forming a stable suspension therein. The magnetic parti- between the two ends. The bames are dimensioned to cles typically comprise a receptor capable of specific mtrict the cross-sectional area of the passageway in binding to a target substance of interest in the test me- which the test medium flows through the container to a dium. If it is desired to separate target substances from region surrounding the axis. The baffles are preferably test medium in a steady state, a suitable container hold- inclined radially downwardly along the direction of 55 ing the test medium and the receptor-magnetic particle flow, to guide toward the wall any magnetically respbn- conjugates are placed in the magnetic separator for sive colloidal particles coming into contact therewith. batch-wise processing. The external magnetic means The conduit preferably has laterally spaced apart outlet disposed around the container produces a magnetic means disposed at the end opposite the inlet. One outlet field gradient in the test medium, which causes the means may be provided along the periphery of the out- 60 magnetic particles to move toward the wall and to let end of the container for collecting the magnetic become adhered thereto. particles. Another outlet means is centrally positioned In the method of the invention which employs a at the outlet end for discharging the test medium. plurality of containers held in a M e r , the magnetic

In a particularly preferred embodiment, the magnetic field gradient causes the magnetically responsive colloi- field generating means may comprise sets of four or six 65 dal particles in the test medium to move toward and permanent magnets or electro-magnets. The magnets adhere to the wall of each container closest to the mag are arranged so as to define a cavity which accommo- netic means. In accordance with this method, the orien- dates the container. In this embodiment, the polarity tation of the wall of each container in the carrier rela-

5,186,827 7 8

tive to the magnetic means may be controllable to cause the particles to adhere more uniformly around the wall of each container.

In another embodiment of the method of the invention, the test medium being separated may be flowed 5 through the separator. The magnetic field gradient intensifying means produces an "open" field gradient of sufficient strength to pull the magnetic particles from the test medium moving at a predetermined rate and to adhere them to the wall. The non-magnetic test medium 10 illustration; k discharged from the contaher at the outlet end. In a related embodiment of this method, in which the container includes one Or more bames, the test medium to be separated is poured into the inlet opening at one end of the conduit. As the test medium moves through the 15 nets; conduit, the magnetic particles in the test medium are attracted by the magnetic means toward the Wall of the conduit and thereby comes in contact with the bames. The baffles are arranged to cause the particles to be camed toward the wall of the separation vessel. The 20 ment of the invention in which a flow.through magnetic means may be operable to cause the particles to become adhered to the interior wall of the separation vessel, or to permit particles to move down the wall for collection at one or more outlets provided along the

test medium may be removed at an outlet laterally spaced from the particle outlet(s) in the center portion DESCRIPTION OF PREFERRED of the conduit at the end opposite the inlet end. EMBODIMENTS

In carrying out the methods of the invention, the Preferred embodiments of the present invention and non-magnetic test medium may be removed from the 30 separator while the magnetic particles are on methods will now be described in detail with reference the walls of the container and subjected to further pro- to the drawings'

bjospecific reactions in this way, resuspension of the present invention have particular utility in various the magnetic particles bearing the target is 35 laboratory and clinical procedures involving biospecific effectively obviated Accordingly, this method substan- afinity reactions. In such procedures, particles are used

the cost of, bioanalytical testing. (i.e., particles which are superparamagnetic and capable of remaining in suspension in a non-magnetic test me-

present invention, that in performing the separation 40 dium), and which comprise a receptor capable of bind- methods described herein, there is no need ing the substance of interest in the test sample. In the certain reagents present in excess in the test medium present method, after the receptor binds the target sub- before addition of a magnetic common capture agent, stance, the magnetic separator removes the magnetic such as colloidal magnetic goat antimouse Fc particles. Panicles from the test medium via €IGMS- This discovery may be used to practical advantage in 45 Such biospecific affinity reactions may be employed that obviating removal of unbound labelling mono. in testing biological Samples for the determination Of a clonal antibody, for example, not only simplifies separa. wide range of target Substance, representative of which tion procedures generally, but particularly enhances are cells, cell ComPonenU, cell S'JbPoP'Jlations @oth cell viability in bioanalytical procedures performed on eukaryotic and prokaryotic), bacteria, parasites. anti- cells, e.g., cell separations of clinical interest. In this SO gens, specific antibodies, specific biological factors, connection, the present invention also provide a such vitamins, viruses and specific nucleic acid se- method for separating a membrane-contahing bioen- quences, as in the case of gene probe analysis. Thus, the tity, having a characteristic determinant, from a test magnetic separation apparatus and methods Of the in- sample by the steps of: introducing into the test sample vention may be used to carry Out cell separations for the a receptor having binding specificity for the determi- 55 analysis or isolation of cells including, by way of exam- nant, in an amount sufficient to bind the receptor to the ple: T-cells from a T-cell lymphoma cell line; B-cells determinant and to provide excess receptor in the test from a B a l l lymphoma cell line; CD4 positive cells sample; introducing into the test sample, in the presence from leukocytes; lymphocytes from leukocytes; tumor of the excess receptor, a multivalent capture agent cells from normal cells; and stem cells from bone mar- which binds to the determinant-bound receptor, 60 row cells. thereby to form a bioentity-receptorcapture agent The methods of the invention may also be used for complex, at least one of the determinant-bound receptor immunospecific isolation of monocytes, granulocytes and the capture agent having associated therewith col- and other cell types; removal of rare cells; depletion of loidal magnetic particles; and separating the complex natural killer cells; determination of reticulocytes; and from the test sample under the influence of a high gradi- 65 assays for neutrophil function, e.g., for determining ent magnetic field. changes in membrane potential, performing oxidative

From the foregoing summary, it will be appreciated burst analysis, phagocytosis /$says and opsonization that the present invention provides separation apparatus studies.

and methods of relatively simply construction and operation which enable the efficient and effective separation of target substance-bearing magnetic particles from a test medium.

DESCRIF'T1oN OF THE DRAW*NGS FIG. 1 is a perspective view of a magnetic separation

apparatus embodying the present invention, portions of the apparatus being broken away for the purposes of

FIG. 2 is a plan view ofthe apparatus shown in FIG.

FIG. 3 is a plan view of a related embodiment of the comprising six mag-

FIG, 4 is a fragmentary perspective view of a related of the invention in which severd conlain.

F ~ G . 5 is a view in perspective of another embodi-

FIG. is a vertical section through the apparatus of

Like characters of reference designate parts in

1;

invention having a magnetic

ers are held in a c a ~ e r ;

container is provided with

FIG,

barnes;

periphery ofthe wall at the end opposite the inlet. The 25 those figures of the drawings in which they occur,

cessing, as desired. By perfombg analyses involving The magnetic separation apparatus and methods Of

tially reduces the processing time required for, and thus which are at Once magnetically responsive and

It has also been discovered, in accordance with the

5,186,827 9

Similarly, the present magnetic separation apparatus and method may be used in bacterial or parasite separation or analysis, including the separation of various bacteria and parasites from fecal matter, urine, sludges, slumes and water (e.g., ground water or streams). The present invention may also be used in separating various bacteria in food products (liquids to solids) sputum and urine.

The preferred magnetic particles for use in carrying out this invention are particles that behave as true colloids. Such particles are characterized by their sub- micron particle size, which is generally less than about 200 nanometen (nm.) (0.20 microns) and their stability to gravitational separation from solution for extended periods of time. Suitable materials are composed of a crystalline core of superparamagnetic material surrounded by molecules which may be physically absorbed or covalently attached to the magnetic core and which confer stabibing colloidd properties. The size of the colloidal particles is sufficiently small that they do not contain a complete magnetic domain, and their Brownian energy exceeds their magnetic moment. As a consequence, North Pole, South Pole alignment and subsequent mutual attraction/repulsion of these colloidal magnetic particles does not appear to occur even in moderately strong magnetic fields, which contributes to their solution stability. Accordingly, colloidal magnetic particles are not readily separable from solution as such even with powerful electromagnets, but instead require a relatively high gradient magnetic field to be generated within the test medium in which the particles are suspended in order to achieve separation of the discrete particles.

Magnetic particles having the above-described properties can be prepared as described in U.S. Pat. No. 4,795,698, the entire disclosure of which is incorporated by reference in the present specification, as if set forth herein in full.

For cell separations, the test medium is typically prepared from appropriately prepared body fluids, such as blood, urine, sputum or secretions. It is preferable to add the colloidal magnetic particles to the test medium in a buffer solution. A suitable buffer solution for this purpose comprises a mixture of 5% bovine serum albumin ("BSA") and 95% of a biocompatible phosphate salt solution, optionally including relatively minor amounts of dextrose, sodium chloride and potassium chloride. The buffer solution should be isotonic, with a pH about 7. The protein serves to decrease interactions which tend to interfere with the analysis. The target substance may be added to the test medium before, after or simultaneously with introduction of the magnetic particles. The methods of the invention take advantage of the diffusion controlled solution kinetics of the colloid magnetic particles, which may be further enhanced by the addition of heat to the test medium. The test medium is usually incubated to promote binding between the receptor and any ligand of interest present therein. Incubation is typically conducted at room temperature or at a temperature slightly above the freezing point of the test medium (i.e., 4' C.). Occasionally, incubation can be camed out at 37' C. The period of incubation is normally of relatively short duration (Le., about 2-15 minutes). The test medium may be agitated or stirred during the incubation period to facilitate contact between the receptor and ligand.

If a small percentage of the buffer solution is replaced by a suitable anionic polyelectrolyte, binding of the

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10 receptor to a material other than the target substance in the test medium (;.e., non-specific binding) is noticeably reduced. Satisfactory results have been obtained using a commercia1 scale inhibitor sold under the name Tamol 850, which is available from Rohm and Haas, Philadel- phia, Pa. Tamol 850 is sold as an aqueous solution of polymethacrylic acid, having a molecular weight of 12,ooO (weight average), total solids of 29-31%, density of 9.9 Ibs./gal. (at 25" C.), a Brookfield viscosity of 125-325 (at 25') and a spindle/speed of #2 @60. The addition of about 0.1% to about 3% Tam01 850 (on an active basis) to the phosphate buffer will generally be adequate to reduce non-specific binding in the practice of the invention.

In carrying out the method of the invention in which various bioentities are magnetically separated from a test medium containing excess receptor, antibody which binds selectively to the bioentity of interest is generally used as the receptor. Monoclonal antibody is preferably used for this purpose. However, non-antibody receptors, including antigen for antibody-producing cells or antigen processing cells, lectins, such as concanavilin A, soybean agglutinin, wheat germ agglutinin and the like, biotin-labeled reagents or hapten- labeled reagents, may be used, if desired.

The capture agent is a substance capable of binding selectively to the receptor, so as to form a bioentity- receptor-capture agent complex. Suitable capture agents include Protein A or Protein G, where Ig is used as the receptor; avidin, where a biotin-labeled reagent is used as the receptor; and anti-hapten, where a hapten- labeled reagent is used as the o receptor. Either biotin or a hapten may be used to facilitate capture of lectin receptors, e.g., concanavilin A, soybean, agglutinin or wheat germ agglutinin, which bind selectively to membrane-containing bio-entities comprising carbohydrate or glycoprotein components. Haptedanti-hapten pairs which are suitable for this purpose include dinitrophenol (DNP)/anti-DNP, fluorescein/anti-fluorescein or arsanilic acid/anti-assanilic acid. The capture agent preferably comprises colloidal magnetic particles which enable separation via high gradient magnetic field; however, the receptor may comprise colloidal magnetic particles, if desired.

After binding of the receptor to the substance of interest is allowed to occur, magnetic separation of the colloidal magnetic particles from the test medium is performed using the apparatus and methods of the present invention. The test medium is placed in or passed through a suitable separation vessel for batch-wise or continuous processing, as desired. Magnetic means disposed around the external periphery of the container generate a high gradient magnetic field or flux within the test medium transverse to the walls of the container. The magnetic means comprises a plurality of north magnetic poles and a plurality of south magnetic poles positioned alternatively around the container. In accordance with certain preferred embodiments of the invention, four or six mamets surround the container with

M) equidistant intervalstherebetween, so that the magnetic field is substantially uniform along tbe walls of the container. The high gradient generated in this way causes the magnetic particles to migrate toward the surface of the wall of the container to which the particles adhere,

65 rendering them easily separable from the test medium. FIGS. 1 and 2 illustrate an embodiment of a magnetic

separator in accordance with the present invention. The separator 21 comprises a container 23 having a periph-

5,186,827 11

era1 wall 25 and an open top for receiving the test medium. The magnetic separator also includes four magnets 27 which define a cavity 33 adapted to receive the container. The magnets 27 are equally spaced along the inner surface of cylindrical ferromagnetic housing or yoke 31, on which they are mounted, e.g., by cementing. The magnets may, if desired, be provided with magnetic flux concentrating means, such as pole pieces 29, which permits variation in the lateral dimension of cavity 33 to accommodate containers of different dimensions, or pole pieces which make the field more uniformly radial.

As shown in FIGS. 1 and 2, the container used to hold the test medium is a microtiter well. The container is positioned substantially coaxially with cavity 33 defined by the magnet faces. In that position, the magnetic field in the test medium 35 adjacent to the wall approaches the magnetic field generated by the magnets. In contrast, there is virtually no magnetic field in the part of the container located along the axis 37 of cavity 33, i.e., the test medium most distant from the wall.

The wall of the microtiter well exposed to the test medium provides an ample surface area for adherence of the colloidal magnetic particles. An advantage of the magnetic separator of the invention, when utilized under the conditions described above, is that by appropriately regulating the quantity of magnetic colloid, the particles tend to deposit substantially uniformly upon surfaces in contact with the medium where the magnetic gradient is high. As a result, particles may be caused to be deposited on a broad portion of the internal surface area of the wall in what is effectively a single layer, as opposed to multiple layers or particle agglomerates, which tend to entrap potentially interfering substances, as when formed on a smaller surface, such as occurs in magnetic separators of the prior art. In experiments employing IgG-bearing magnetic colloid, for example, it was found that when colloid mixed with enzyme was collected on the side of a separation vessel, enzyme became trapped as the colloid began to accumulate in layers. In the practice of this invention, by contrast, the colloidal magnetic particles are sufficiently thinly deposited on the container surface that there is virtually no entrapment of potentially interfering substances. To this end, it is preferable that the portion of the container wall surface in contact with the test medium be selected so that the wall’s aggregate collecting surface area is greater, by a factor of about 2, than the surface area that wold be occupied by all of the magnetic particles in the test medium, if deposited in a substantially continuous single layer.

For permanent magnet devices, the magnetic field strength of the external magnetic means (magnets 27 in FIG. 2) at the pole faces should be in a range of 4-10 KGauss, and more preferably between about 6-8 KGauss. The preferred distance between each magnet and the container shown in FIGS. 1 and 2 is generally about 0.1 cm. to about 2.0 cm. with the most preferable distance being about 0.5 cm. The field strength of the external magnet or magnets should be great enough, and the distance between the magnets and the container 23 for the test medium short enough to give eficient separation of the magnetic particles. With electromagnets, considerably higher field strengths at the pole faces, on the order of 15-30 KGauss, can be achieved. In this way, very high gradient fields are obtained.

The cavity 33 should be formed by the magnets with sufficient excess space for manipulation of the container

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12 23. For example, an elevating mechanism (now shown) may be positioned in the cavity to raise and support the container 23 in the position illustrated.

The pole pieces 29 illustrated in FIGS. 1 and 2 may be fabricated of any magnetizable material. The pole pieces are shown in the form of magnetized bodies of trapezoidal cross-section, but may be fabricated in other shapes, such as triangular cross-section. These configurations aid in concentrating the magnetic flux emanating from magnets 27. The pole pieces 29 may be held in place on the faces of magnets 27 by magnetic attraction. Alternatively, magnets 27 may be fabricated such that the pole piece is unitary with the body of the magnet.

FIG. 3 illustrates another embodiment of the magnetic separator of the invention that is similar to the magnetic separator shown in FIGS. 1 and 2. The separator 121 shown in FIG. 3 comprises a container 123 in the form of a cylindrical tube or the like. The container may be closed at one end, e.g., a test tube for batch-wise processing, or open at both ends, e.g., a capillary or larger diameter tube for continuous processing. The container has a wall 125 and an opening for receiving the test medium being separated. The separator also comprises magnetic means represented by six magnets 127 which are equally spaced around the container. FIG. 4 illustrates a variation of the magnetic separa-

tor of the invention illustrated in FIGS. 1 and 2. In FIG. 4, the separator 221 comprises a plurality of containers 223 held bv a camer 241 and a mametic means external

30 to the carker. Each container has; peripheral wall 225 and an open top. The magnetic means comprises four magnets 227 having curved faces disposed concentrically around the camer.

The carrier 241 comprises a generally planar non- 35 magnetic base 243, with separate non-magnetic com-

partments 253 fitted into the base at an outer edge thereof. Each such compartment is dimensioned to hold securely one of the containers 223 in an upright position.

FIGS. 5 and 6 illustrate another embodiment of the magnetic separator of the invention. The magnetic separator 321 comprises a cylindrical container 323 with openings at either end to permit the test medium being separated to flow through the container. The separator

45 also comprises four magnets 327 which are spaced around the container with substantially equal intervals therebetween.

The container illustrated in FIGS. 5 and 6 has a peripheral wall 325 that defines an inner cross-sectional

SO space 362 inside the container. One end of the container has an inlet port 363 that is dimensioned to permit entry of test medium to the central portion 365 of the inner cross-sectional space between the wall and the central portion. The baffles are also inclined downwardly along

55 the direction of flow toward wall 325. Hence, in passing through the container, the magnetically responsive colloidal particles are attracted by the magnets 327 onto the baffles and toward the wall. The baffles guide the particles along to a point at which the field gradient

60 becomes sufficiently strong to cause the particles to adhere to the wall. The container also has at least two outlets. One outlet 369 is along the periphery of the wall for collection of the particles. A second outlet 371 is aligned with the axis of the container for discharge of

The magnetic particles may be separated from the test medium using the magnetic separator apparatus described above, after the test medium has been sub-

40 .

65 the non-magnetic test medium.

5186,827 - 3 -

13 jected to the magnetic field for a sufficient time to cause the magnetic particles to migrate and adhere to the container wall. The non-magnetic components of the test media may be removed by decantation or aspiration, with the container still in the separator. A buffer, a wash liquid, or the like may then be added, while the container remains within the separator, to contact the wall for washing the adhering magnetic particles substantially free of any residual non-magnetic components. If desired, the wash liquid may be removed and the process repeated. If it is advantageous to resuspend the magnetic particles, the container may be removed from the magnetic field and manipulated to allow the magnetic particles to be dislodged from the walls and resuspended in a suitable liquid medium, e.g., to facilitate analysis.

The magnetic separation apparatus and methods of the invention permit advantageous use of diffusion controlled solution kinetics of the primary incubation mixture. Moreover, various analytical procedures, including quantitative determinations, may be performed on the magneticaIly immobiiized colloid. Such steps include washes for removal of non-specifically bound substances, secondary immunochemical reactions or detection reactions (e.g., enzymatic, fluorescent or chemiluminescent reactions).

The ability to retain the magnetic particles adhered to the wall of the container after the test medium has been removed is of considerable utility. Certain operations are more efficiently carried out in this way, such as washing or rinsing the target substance, e.g., cells or labeled components of a reaction mixture, while avoiding a separate resuspension step. In addition, secondary reactions such as those involving the interaction of labeled immunoreactive agents with a target substance carried by the magnetic particles may be performed more efficiently with the particles adhered to the wall. Here again, resuspension of the colloidal magnetic particles is avoidable. Furthermore, in performing enzyme- labelled immunoassays in accordance with the present invention, substrate incubation is preferably carried out directly on the colloidal magnetic particles immobilized in the separation apparatus.

Performing the magnetic separation method of the invention bstchwise, i.e. in a steady-state system, as described above, instead of in a flow through system, has certain advantages. Immobilized magnetic particles bearing the target substances are not dislodged due to collisions with other particles. Moreover, batchwise operation eliminates dislodgment of immobilized magnetic particles due to shear forces produced by a flow- inn test medium. In other words, the adherence of the

14 In general, the magnetic particles are relatively easily

separated from the container wall after removal from the magnetic field. The particles may be dislodged by contacting the wall with a modified buffer solution, or a

5 bath sonicator. Alternatively, the particles may be collected as they are dislodged from the wall with a probe sonicator.

. Multipole magnetic separation devices such as those described above in which near neighbor pole faces are

10 of opposite polarity create high gradient fields wherein the magnitude of the gradient is limited only by the field density at the pole face and the distance separating opposing poles. Compared with gradient fields produced by inducing magnetic fields on ferromagnetic

15 material (fine wires, rods, spheres etc.) placed in external fields, the multipole devices of the invention have substantial advantage because the field strengths at the center of the opposing poles is zero. Hence, the gradients produced are only limited by the ability to produce

The following examples further describe in some detail the manner and process of making and using the present invention and set forth the best mode contemplated by the inventors for carrying out the invention,

25 but are not to be construed as limiting the invention. All temperatures given in the examples are in C., unless otherwise indicated.

20 fields greater than zero.

EXAMPLE 1

To demonstrate the utility of high gradient fields produced by multipole apparatus in effecting magnetic separation in accordance with the present invention, quadrupole and hexapole devices were constructed and tested. For this demonstration, permanent rare earth

35 magnets were used (Crumax 355, Crucible Magnetics, Elizabethtown, Ky.); however, electromagnets could also be used, if desired. Quadrupole and hexapole devices similar to those shown FIGS. 1-3 were made comprising a yoke in the form of a 5 cm. long cylindri-

40 cal steel pipe of 2.5 inch internal diameter (obtainable from plumbing supply distributors) and bar magnets disposed on the inner walls of the cylinder. Crumax 355 bar magnets 1.27 X 1.27 cm. by 5 cm. (magnetized through one of the 1.27 cm. dimensions) were mounted

45 on the inner surfaces of the cylinder, with the long dimension of the bar magnet parallel to the axis of the cylinder. For the quadrupole apparatus, four bar magnets were employed, placed at 9, 180', 270" and 360' with respect to a plane normal to the axis of the cylin-

50 der. The hexapole apparatus was similarly constructed except that six bar magnets were placed at W', 120", 180'. 240". 300" and 360" around a cylindrical yoke. In

30

magnetic particles to the wall is sumciently strong to both the quadrupole and hexapole apparatus, the polar- permit washing, secondary reactions, and interactions ity of the magnets alternated around the yoke. For the with other reagents to occur without appreciable dis- 55 quadrupole and hexapole apparatus thus constructed, lodgment of the magnetic particles from the wall. In opposing magnet faces were 3.4 cm. apart (pole face addition, the adherence of the magnetic particles to the gap radius= 1.7 cm) and the field density at each pole container wall is maintsined to some extent even if the face measured 5 5 KGauss using a Gauss meter (Ap- container is removed from the magnetic field before plied Magnetics Laboratory, Baltimore, Md.). As the further reaction with, or treatment of the particles. 60 field at the center was 0 the average field gradient was

Nevertheless, in certain applications it is very desir- 3.2 KGauss/cm. able to be able to employ a magnetic separator adapted To evaluate the effectiveness of the quadrupole and for use in a flow through process. For example, flow hexapole apparatus for radially separating small mag- through processes are very desirable for separating netic particles or cells to which magnetic particles are large quantities of material of a uniform type. Surpris- 65 bound, the following experiments were done. A bovine ingly, the separator of the invention avoids the problem serum albumin coated magnetic colloidal (60 nm. diam- of dislodged particles noted above that commonly oc- eter partides) was prepared by the method described in c u m when employing a flow through process. Example 1 of Owen et al., U.S. Pat. No. 4,795,698. A

5,186,827 15 16

small agglomerate (200-300 nm) of this magnetic col- EXAMPLE 2 loid was formed by the addition of NaCl which causes a partial saltingsut of the colloid, as described in co- Mammalian cells were separated using the multipole pending US. patent application Ser. No. 389,697, filed apparatus of Example 1, above. The target substance in Aug. 4, 1989. Such agglomerates have magnetic separa- 5 this experiment was human T-cells (ATCC Accession tion properties similar to cells to which are bound mag- NO. CCLl19 CCRF-CEM) which were maintained in netjc colloids bearing specific cell surface antibodies. cell culture under conditions available from ATCC. Hence, the preparation was useful for assessing how cells were either labeled With chromium 51 (CR'') by cells would separate in a multipole high gradient field the Procedure of GGV Klaus* LymPhocYres* Prucfico' apparatus. This agglomerate is also useful for evaluating lo A P P ~ C ~ ~ IRL Press Ltd.9 P. 144 (*987)9 or they were separability of the ColIoid, but in a more reasonable fluoreantly lheled with acridine orange according to period of time, as compared with separation of the ac- a procedure known in the art* After labeling by tual colloid. using 250 ul aliquots of the above- either procedure, cells were washed in the usual man- dficri&d agglomerate in single microtiter wells (0.7 ner. cells were resuspended to approximately 3 x 106

ered saline solution containing 0. I % sodium azide, pH 7.2, supplemented with 1% (w/v) BSA. A dextran devices were determined by placement of the wells at

different locations within the field and observing the coated colloid nm. diameter) which was made by collection of agglomerate on the wall of the microtiter the Molday procedure, as described in u,s. Pat. No,

minutes). From these experiments, it was found that for to cells, was used to

are radial and substantially axisymmetric over a radius suspension.

tions performed at greater radii showed that magnetic 25 glass cylindricaf vessels of 1-76, 2.38 and 3-17 cm. i.d., agglomerate Was not collected on the Outer radii ofthe respectively. These vessels were placed in the center of microtiter wall in a unifom manner1 whereas highly the quadrupole or hexapole separation apparatus which uniform radial collection was observed at the lesser were mounted on the stage of an inverted microscope radii. 3o (Zeiss Axiovert 35 MI Thornwood, N.Y.) in such a way

To further substantiate the axisymmetry of collection that separation in all parts of the vessel was observable within the radial regions described immediately above, via fluorescent microscopy of the cells. For the two an open-top cylindrical glass vessel of 2 cm. internal smaller diameter radii vessels employed, magnetically diameter containing the above described magnetic ag- induced migration of the cells to the peripheral wall of glomerate was placed in the center of the quadrupole or 3s the vessel was completely uniform. When the 3.17 cm. hexapole devices and separation observed. This was diameter vessel was used, irregularities of collection done at several dilutions of the agglomerate so that were observed on the periphery at locations corre- deposits of magnetic particles on the walls of the vessel sponding to the gaps between near neighbor magnets. could be observed at several densities. In all cases, the Thus, for the quadrupole apparatus there were four deposits on the walls were observed to be substantially 40 regions (go', 1 Bo", 270' and 360") where excessive COl- uniform. When separation was performed in a vessel lection of cells occurred; and with the hexapole six such placed substantially off-center in the apparatus, non- regions were observed. Hence, by selecting a separation uniformity Of collection was observed on the cylinder vessel of appropriate dimensions for a given separation

apparatus, substantially uniform deposition of the mag- wall closest to the pole faces. may be

would separate similarly, a dextran coated colloid (80 achieved. nm diameter), prepared as described in US. Pat. EXAMPLE 3 4,452,773 to Molday, was placed in the glass vessel

by which time a uniform deposit was Obtained On the walls of the vessel. It is thus reasonable to expect

i.d,) the local magnetic fields within these multipole Is d s / d in a cell-compatible isotonic phosphate buff-

well after clearing from the test medium (generally 3-5 20 4,452,773, and showed very high non-specific binding

the quadrupole and hexapore apparatus the concentration of the colloid was 17 ug iron/mi, of cell cells, The

forces

Of about of the pole face gap radius- Separa- Fluorescently labeled -11s were placed in opentop

To demonstrate that a non-agglomerated colloid 45 netic particles on the Of the

To determine how efficiently cells can be induced to

(where the field is zero), radio-labeled cells were placed in a 2.38 cm. i,d. cylindrical vessel which was then

described above and to separate for Io min- ~~ radially from the center of a multipole device

that col'oidal particles are %parable placed at the center of the hexapole d e i c e constructed apparatus Of the type described above* in which the as previously described. AliquoB of the *St medium gradient the 55 were carefully removed periodically from the e n t e r of

of cells from this region was calculated. By three min-

are higher than those generated present experiment. the vessel and counted for CRsl, from which depletion

ratus was measured to be zero* ma@etic particles were utes, 30% of the cells were removed from the centcal collected from the center Of the Vessel. Thus, it appears region, by Seven minutes 77% of the cells were re- that the thermal energy of small magnetic Particles in 60 moved and by twelve minutes 86% removal was ob- combination with ~~g due to thermal influences h sewed, Thus, even in the c s e of cells which exhibit the solvent are sufficient to cause diffusion to the high essentially no thermally induced motion there is sufi-

Although the magnetic field at the center ofthe appa-

gradient region of the vessel. Field uniformity and cient mixing of cells such that they can be pulled from added axisymmetry can theoretically be achieved by the very center 'of such devices. This experiment dem- appropriate cuwiture of the pole faces and magnets 6s onstrates that cells flowing through a cylindrically used to construct multipole devices. By minimizing the shaped container placed in a multipole apparatus are gap between near neighbor magnets further reduction removable in an efficient manner. Using appropriate in field irregularities near these regions can be achieved. flow parameters and with appropriate baming a multi-

5,186,827 17

pole device could be used for continuous removal of cells.

EXAMPLE 4

A quadrupole separation apparatus was constructed from a cylindrical steel yoke having 5.78 and 8.32 cm. inner and outer diameter, respectively, and measuring 5.10 centimeter high, and 4 Crumax 355 permanent bar magnets of dimensions0.635 X 2.54 x 5.10cm., magnetized through the 2.54 cm. dimension. The four magnets were placed on the inner surface of the cylinder at 90", 180", 270" and 360' and aligned such that the 5.10 cm. dimension of each bar magnet was parallel to the axis of the yoke and the 2.54 cm. dimension was radially aligned. As with the above described quadrupoles, opposing magnet faces were of the same polarity and near neighbor magnets had opposite polarities at their faces. To maintain the bar magnets properly aligned, i.e., at 90" to each other, arcuate spacer elements made of aluminum were machined and fitted into the spaces bctween bar magnets. This arrangement created a quadrupole having a gap between opposing pole faces of 0.70 cm. Using a Gauss meter, the field at the pole face surface was measured at 5.95 KGauss and zero at the center. Thus, the average field gradient for this assembly was 17 KGausshm. To determine how well this separation apparatus removed colloidal sized magnetic particles from solution, a 6 cm. length of glass tubing, 0.30 cm. inner diameter, was inserted into the quadrupole along its axis. This tube was connected to a syrine pump (Harvard Apparatus, Dedham, Ma.) such that test medium could be introduced into the glass tubing positioned upright in the field of the quadrupole and subsequently removed for inspection. Bovine serum albumin- coated colloidal magnetic particles of 80 nm. diameter, made according to Example 1 of Owen et al., US. Pat. No. 4,195,698, and suspended in 20 mM phosphate buffer, pH 7.5, were drawn up into the glass tubing, positioned in the magnetic field and separation was allowed to take place. When suspensions of this colloidal material containing from 20-100 u g h 1 of iron were subjected to the magnetic field gradient,'complete separation occurred in about 6 minutes, as determined by spectrophotometric examination of the solution pumped out of the separation tube.

To determine the distribution of the colloidal materia1 on the inner surface of the separation tube, levels of colloidal material similar to the above were separated, after which the mother liquor was pumped from the tube using the syringe pump and the magnetically deposited material was allowed to dry on the inner surfaces of the tube. Deposited material so dried was thereafter not affected by magnetic gradient. From inspection of these tubes, it was apparent that material is evenly deposited on the inner surfaces of the separation tubes.

EXAMPLE 5 Typically, when cells are immune specifically re-

trieved by means of labelling with cell-specific niono- clonal antibody (MAb), it is customary to incubate cells with MAb, wash out excess MAb and next incubate with a multivalent common capture agent, e.g., second antibody or avidin on a suitable solid support, such as magnetic particles. The purpose of removing excess MAb is to prevent agglutination between non-cell bound MAb and the multivalent common capture agent, and also to preserve binding sites on the common

5

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35

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50

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18 capture agent. To demonstrate the efficiency of external gradient field devices for performing immune specific cell separations, while at the same time obviating removal of excess MAb before the addition of common capture magnetic colloid, the following experiments were done. Afinity purified goat anti-mouse Fc (GAMFc) (Jackson Immunoresearch Lab, West Grove, Pa.) was coated along with bovine serum albumin (BSA) onto colloidal magnetic particles as described in U.S. Pat. No. 4,795,698 for use as the common capture agent. The final concentration of GAMFc and BSA on the colloid were 0.7 mg and 0.05 mg, respectively, per milliliter of final colloid. CEM cells were Crs*-labelled and resuspended to a final concentration of 2.5 X 1@/rnL in PES as described in Example 2 above. To 1.35 mL of cell suspension, 85 uL of anti-CD 4 MAb (Gen-Trac, Plymouth Meeting, Pa.) was added at a concentration of 0.1 rng/mL (total 8.5 micrograms MAb) with mixing and incubated five minutes. Next, 1.35 mL GAMFc magnetic colloid was added (67.5 micrograms Fe), mined, and incubated for five minutes; 2.6 mL of this mixture was immediately placed in a fabricated polystyrene separation vessel (internal dimensions 10 mm x 8 mm X51 mm high). Finally, this separation vessel cell was placed in a quadrupole device.

The quadrupole was constructed from four bar magnets which were 5.0 cm long, 1.6 cm wide and 1.9 cm deep and they were magnetized through the 1.9 cm dimension. The flux density at the pole faces was 6.6 KGauss and the radius of the quadrupole was 1.1 cm. Thus, the gradient for the device so constructed was 6 KGauss/cm. The 10 x 51 mm face of the separator vessel was placed directly on one of the pole faces, hence separation took place radially through the 8 mm dimension of the vessel.

Magnetic separations were performed for five minutes, ten and fifteen minutes, respectively, on identically prepared samples. All operations were performed at room temperature.

Further, the GAMFc colloid buffer (PBS containing 5% BSA) also included 1% Tamol-850 (Rohm-Haas, Philadelphia, Pa.) to prevent non-specific binding of colloid to cells. Cell supernatant was removed from the separation vessel with a 20 gauge needle placed at the back of the vessel (nearest the center of the quadrupole) and aliquots counted for Crs', and also visually counted on a hemocytometer. For these experiments, it was found that 48,83 and 90% of the cells were pulled to the surface of the vessel facing the pole piece in 5, 10 and 15 minutes, respectively. In the absence of MAb, or with non-specific MAb no depletion of cells took place. For experiments done in which cells were washed free of excess MAb after MAb incubation, followed by addition of GAMFc colloid, cell depletions over the 5, 10 and 15 minute magnetic separation periods were considerably lower.

It is interesting to note that the amount of MAb left free in solution far exceeds that on cells. If it is assumed that the cell has 50,OOO receptors ana can bind that many MAbs, then less than 0.05 micrograms of MAb is consumed, or 8.45 micrograms remain in solution. De- spite that quantity of MAb in solution and the fact that there is excess colloid in solution, separation is readily accomplished. At the dilution of GAMFc colloid used for cell separation, agglutination reactions between MAb remaining free @ solution and excess magnetic capture agent are not visually apparent and, indeed, if

5,186,827 19

small complexes do exist, they are not deleterious to the separation.

The ability to do such separations without removing excess MAb has very beneficial implications for cell analysis, in addition to simplifying procedures. Not only would improved viability of cells be expeted by reducing manipulations, but obviating removal of excess MAb permits the use and exploitation of lower afinity antibodies which, when added in excess, can be "forced" on to receptors, but which readily disassociate when excesses are removed. Additionally, this methodology enables the use of relatively low-affinity, non- antibody receptors including lectins, such as concanavilin A, soybean agglutinin or wheat germ agglutinin for cell labeling via selection of an appropriate common capture magnetic colloid.

From the foregoing experiments, it is clear that a multipole arrangement is useful for separating small magnetic particles from a fluid containing same, e.g., for analytical purposes. Alternatively, a continuous flow system of essentially the same design could be constructed for removing colloidal magnetic material from a moving carried fluid, where such capability is desired in a particular application, such as bioprocessing.

While various aspects of the present invention have been described and exemplified above in terms of certain preferred embodiments, various other embodiments may be apparent to those skilled in the art. For example, the method of the invention may be scaled up to accommodate large volumes of material for various industrial

20 4. A magnetic separator as claimed in claim 1,

wherein said magnetic means comprises multiple magnets.

5. A magnetic separator as claimed in claim 4, 5 wherein said container is of cylindrical configuration

and each of said magnets has a curved pole face, said faces being disposed in a circle substantially concentric with said container.

6. A magnetic separator as claimed in claim 4, 10 wherein said magnetic means also comprises a magnetic

nux concentrating means associated with at least one of said magnets.

7. A magnetic separator as claimed in claim 6, wherein said flux concentrating means is a pole piece.

8. A magnetic separator as claimed in claim 1, wherein said magnetic means comprises at least four magnets surrounding said container with poles of like polarity confronting one another.

9. A magnetic separator as claimed in claim 1, 2o wherein said magnetic means comprises six magnets

spaced substantially equidistantly around the periphery of said container. IO. A magnetic separator useful for separating mag-

netic particles from a non-magnetic test medium in 25 which said particles are suspended, said separator com-

prising: (a) a plurality of non-magnetic containers, having

peripheral walls with internal surface area for re-

cbl a non-magnetic carrier for said ~luralitv of con-

Is

3i ceiving said test medium; ..

processing applications, especially bioprocessing. The invention is, therefore, not limited to the embodiments specifically described and exemplified, but is capable'of variation and modification without departing from the 35 spirit of the invention, the full scope of which is delineated by the appended claims.

tainers, saidcarrier including a bas;, said dase being generally planar and having an outer edge with means for engaging said containers adjacent to said outer edge of said carrier; and

(c) magnetic means surrounding said camer for generating a magnetic field gradient that is operative

What is claimed is: upon magnetic particles within said test medium in I. A magnetic separator for separating magnetic par- each container to attract said particles toward said

ticles from a non-magnetic test medium in which said a internal surface area closest to said magnetic magnetic particles are suspended, the separator com- means, thereby causing such particles to be ad- prising: hered to said surface without substantial entrap-

(a) a non-magnetic container having a peripheral wall ment of interfering substances. with an internal surface area for receiving said test 11. A magnetic separator as claimed in claim 10, medium; and 45 wherein said magnetic means comprises at least four

(b) magnetic means for generating a magnetic field magnets spaced around said carrier, each of said mag- gradient within said container, in which the magnets having a face confronting the face of one other netic field is stronger in the test medium along said magnet, said confronting faces being substantially dia- internal surface area of said wall than in said test metrically opposed and being of the same polarity. said medium most distant from said wall and is opera- 50 containers being positioned between the confronting tive upon said magnetic particles within said test magnet faces. medium to attract said magnetic particles toward 12. A magnetic separator as claimed in claim 11, said surface area and cause such particles to be wherein said magnetic means also comprises a magnetic adhered to said area, said magnetic means compris- flux concentrating means associated with each of said ing a plurality of north magnetic poles and a plural- 55 four magnets. ity of south magnetic poles positioned alternately 13. A magnetic separator 8s claimed in claim 10, around an exterior surface of said peripheral wall wherein said magnetic means comprises six magnets of said container and defining a receptacle for said spaced around said carrier, each of said magnets having container, said container being removably a face confronting the face of one other magnet, said mounted in said receptacle. 60 confronting faces being substantially diametrically op-

posed and being of opposite polarity, said containers being positioned between said confronting magnets.

14. A magnetic separator as claimed in claim 13, wherein said magnetic means also comprises a magnetic

65 flux concentrating means associated with each of said six magnets.

15. A magnetic separator useful for separating magnetic particles from a non-magnetic test medium in

2. A magnetic separator as claimed in claim 1, wherein said magnetic poles are mounted on a cylindrical ferromagnetic yoke disposed around said container, said magnetic poles being intermediate said container and said yoke.

3. A magnetic separator as claimed in claim 2, wherein said magnetic means generates a field of about 5 lo about 30 KGauss.

21 5,186,827

22 which said particles are suspen-Jd, said separator com- (c) saiL container being removi-ly mounted to said prising: magnetic means to permit removal of said magnetic

(a) a non-magnetic container having a peripheral wall field and said magnetic field gradient and discharge forming an internal surface area and an inner cross- of said magnetic particles from said internal surface sectional space inside the container, an inlet port at 5 area by gravitational force. one end dimensioned to cause the test medium to 16. The. separator as claimed in claim 15 wherein the flow into the inner cross-sectional space, and an shape of said cross-sectional space is generally cylindri- outlet port in said container at the end opposite said cal. one end- for discharging said non-magnetic test 17. The separator as claimed in claim 15 wherein said medium; and 10 magnetic means comprises four magnets spaced around

@) magnetic means external to said container and said container with each of said magnets having a face capable of applying a magnetic field transverse to confronting the face of one of the other of said magnets, said peripheral wall thereby to generate a magnetic said faces beiig of the Same polarity, said container field gradient in the test medium within said Con- being positioned between said confronting faces. tainer, in which the magnetic field is stronger in 15 XS. A magnetic separator as claimed in claim 15, said test medium along said internal surface area of wherein said container ,includes at least one bame said wall than in the test medium in a central por- spaced between said ends inside said container and tion of the cross-sectional space, and operative 'transverse to said peripheral wall, said bame having an upon said magnetic particles to attract said parti- upstream surface, a downstream surface and, a central cles toward said surface area and cause such parti- 20 opening to .confine flow of test medium primarily cles to be adhered to said area, said magnetic means through the central portion of said cross-sectional comprising a plurality of north magnetic poles and space, said upstream surface being effective to guide a plurality of south magnetic poles positioned alter- magnetic particles contacting said bame toward said natety around an external surface of said peripheral wall. * * * * * wall of said container; and 25

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CEK'CiYICATE OF EXTLING BY EXPRESS MAIL IIiIXR 37 CFR $1.10 March 25, 1991

NO. OF EXPRESS M A I L LABEL RB34,6115022 DkTE Oh' DEHISIT WInl F'OSl'A!, SERVlCE I hereby a r t i E y tha t t h i s wper or €eo is baing h p s i t e d with the United States Postal Servlca "Express hi1 Post Office to Addressee" serv ice under 57 CFR $1.10 on the da te indicated a b v e , and 1s addressed to the Coinniosioner of patents arid Tradeimrks, Wishitqton DC 20231.

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THE MMEIISSIONER OF PATENTS AND TRADEMARKS Washington, D.C. 20231

Sir:

Inventor(s):

T & d ! t e appl icat ion includes the follouing: ! :$ 3 Number oL Claims [ I-;- Page oC Abstract [xa [q 2 Pages of Declaration and Power oE Attorney [ [

We a r e Liling herewith a United State6 Patent Application oE: Paul A . L i b e r t i , Brian P. Fee:ey and Dhanesh I. Gohel

( T i t l e ) : APPARATUS AND METHODS FOR MAGNETIC SEPARATION FEATURING EXTERNAL MAGNETIC MEANS

Pages o€ s p e c i e i w t i o n

2 Sheet(s) of Drawing ( i n t r i p l i c a t e )

] _- Number of VeriCied Statement(s1 under37 CFH $$1.9 and 1.27 to es tab l i sh Snall Entity S b t u s 3 A- A preliininacy ainandment

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The present invention is directed to magnetic separation apparatus and methods in which magnetic particles are used for isolating substances of interest from a non-magnetic test medium by means of high gradient magnetic separation ( H G M S ) .

BACKGROUND OF THE INVENTION The present invention relates to improvements in

magnetic separators and methods of separation of magnetic particles from non-magnetic media, having particular utility'in various laboratory and clinical procedures involving biospecific affinity reactions. Such reactions are commonly employed in testing biological samples, such as blood or urine, for the determination of a wide range of target substances, especially biological entities such as cells, proteins, nucleic acid sequences, and the like.

As used herein, the term "target substanceu8 refers to any member of a specific binding pair, i.e., a pair of substances or a substance and a structure exhibiting a mutual affinity of interaction and includes such things as cell components,- biospecific ligands and receptors. ItLigandlt is used herein-to refer to substances, such as antigens, haptens and various cell-associated structures, having at least one characteristic determinant or epitope, which are capable of being biospecifically recognized by and Bound to a receptor. llReceptorlu is used herein to refer to any substance or group of substances having a biospecific binding affinity for a given ligand, to

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the substantial exclusion of other substances. Among the receptors determinable via biospecific affinity reactions are antibodies (both polyclonal and monoclonal), antibody fragments, enzymes, nucleic

member of a biospecific binding pair is dependent upon its selective interaction with the other member of the pair.

Various methods are available for determining the above-mentioned target substances based upon complex formation between the substance of interest and its specific binding partner. instance whereby the occurrence or degree of target substance/binding partner complex formation is

5 acids, Clq and the like. The determination of any

10

Means are provided in each

15 determinable. )

In the case of a competitive immunoassay to determine antigen, for example, the antigen of interest in a test sample competes with a known quantity of labelled antigen for a limited quantity of

appropriate reaction period the amount of labelled antigen bound to specific antibody is inversely proportional to the quantity of antigen in the test sample. Competitive assays for antibodies, employing

rather than labeled antigen, function'in an analogous manner. The resulting immune complexes are separated, for example, by immunoabsorption, physico-chemical adsorption or precipitation of either the complexes or

30 unbound antigen. Antibody-bound labeled antigen is then quantified and a standard curve is constructed from known antigen concentrations, from which unknown concentrations of antigen may be determined.

2 0 specific antibody binding sites. Thus, after an

2 5 labeled antibodies (typically monoclonal antibodies)

In contrast, immunometric assays f o r the 35 determination of antigen, commonly known as ttsandwichtt

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assays, involve the use of labeled antibodies instead of labelled analyte. assay, a sandwich is formed in which the ftlayerslg are: antibody/multivalent (minimally bivalent) antigen/antibody.

for each complete sandwich complex (antibody/antigen/antibody) is directly proportional to the amount of target antigenic substance present in the test sample. multi-step fashion with polyclonal antibodies or in fewer steps when monoclonals directed to independent antigenic determinants are employed.

In performing an immunometric

The-amount of the labeled antibody which is bound

Sandwich assays can be performed in

In both the conventional competitive immunoassay and the immunometri)c assay just described, quantification of the target substance requires a physical separation of bound from free labeled ligand or labeled receptor.

Bound/free separations may be accomplished gravitationally, e.g. by settling, or, alternatively, by centrifugation of finely divided particles or beads coupled to the target substance. If desired, such particles or beads may be made magnetic to facilitate the bound/free separation step. Magnetic particles are well known in the art, as is their use in immune and other bio-specific affinity reactions. See, for example, U . S . Patent 4,554,088 and Immunoassays for Clinical Chemistry, pp. 147-162, Hunter et al. eds., Churchill Livingston, Edinborough (1983). Generally, any material which facilitates magnetic or gravitational separation may be employed for this purpose.

Small magnetic particles have proved to be quite useful in analyses involving biospecific affinity reactions, as they are conveniently coated with

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biofunctional polymers, e.g., proteins, provide very high surface areas and give reasonable reaction kinetics. microns have been described in the patent literature, including, by way of example, U . S . Patents N o s . 3 , 9 7 0 , 5 1 8 ; 4 , 0 1 8 , 8 8 6 ; 4 ,230 ,685; 4 , 2 6 7 , 2 3 4 ; 4 , 4 5 2 , 7 7 3 ;

4 , 5 5 4 , 0 8 8 ; and 4 , 6 5 9 , 6 7 8 .

are disclosed to be useful solid supports for immunologic reagents, having reasonably good suspension characteristics when mildly agitated. Insofar as is known, however, absent some degree of agitation, all of the magnetic particles presently in commercial use settle in time and must be resuspended prior to use. employing such reagients.

above, generally fall into two broad categories. The first category includes particles that are permanently magnetized; and the second comprises particles that besome magnetic only when subjected to a magnetic field. The latter are referred to herein as magnetically responsive particles. Materials displaying magnetically responsive behavior are sometimes described as superparamgnetic. However, certain ferromagnetic materials, e.g., magnetic iron oxide, may be characterized as magnetically responsive when the crystal size is about 300A or less in diameter. Larger crystals of ferromagnet-ic materials, by contrast, retain permanent magnet characteristics after exposure to a magnetic field and tend to aggregate thereafter. See P. Robinson et al., Biotech Bioenq. XV: 603-06 (1973) . known. See U . S . Patent No. 4 , 7 9 5 , 6 9 8 to Owen et al., which relates to polymer-coated, sub-micron size

Magnetic particles ranging from 0.7-1 .5

Certain of these particles

This adds another step to any process

Small magnetic particles, such as those mentioned

Magnetically responsive colloidal magnetite is

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magnetite particles that behave as true colloids.

bound-free separations of target substance-bearing magnetic particles from test media will depend on the nature and particle size of the magnetic particle. Micron size ferromagnetic, i.e., permanently magnetized, particles are readily removed from solution by means of commercially available magnetic separation devices. relatively inexpensive permanent magnet located external to a container holding the test medium. Examples of such magnetic separators are the MAIA Magnetic Separator manufactured by Serono Diagnostics, Norwell, MA, the DYNAL MPC-1 manufactured by DYNAL, Inc., Great Neck, New York and the BioMag Separator, manufactured by Advanced Magnetics, Inc., Cambridge, Massachusetts. this type in performing magnetic solid-phase radioimmunoassay is described in L. Hersh et al., Clcnica Chemica Acta, 63: 69-72 (1975). A similar magnetic separator, manufactured by Ciba-Corning Medical Diagnostics, Warnpole, MA is provided with rows of bar magnets arranged in parallel and located at the base of the separator. This device accommodates 6 0

test tubes, with the closed end of each tube fitting into a recess between two of the bar magnets.

system employing cellulose-coated magnetic particles is described in U . S . Patent No. 4,141,687. The automated system exemplified in the '687 patent includes elaborate electromagnetic traps which are operable in a pre-determined sequence by a programmer device under the control of a sample detector.

The above-described magnetic separators have the disadvantage that the magnetic particles attracted

The magnetic separation apparatus/method used for

These devices employ a single

A specific application of a device of

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An automated continuous-flow radioimmunoassay

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toward the magnets tend to form in multiple layers on the inner surface of the sample container where they are entrapped along with impurities that are difficult to remove even with vigorous washing.

Colloidal magnetic materials are not readily separable from-solution as such, even with powerful electro-magnets but, instead, require high gradient field separation techniques. See, R. R. Oder, IEEE Trans. Maqnetics, 12: 428-35 (1976); C. Owen and P. Liberti, Cell Separation: Methods and Selected Amlications, Vol. 5 , Pretlow and Pretlow eds., Academic Press, NY, (1986); J. T. Kemshead and J. Ugelstad, Masnetic Molecular and Cellular Biochem., 67, 11-18 (1985). The gradient fields normally used to filter such materials generate hugh magnetic forces. magnetic separations of colloidal magnetic particles from a test medium, by various manipulations of such particles, e.g., addition of agglomerating agents, is the subject of co-pending and commonly owned U . S .

Patent application Serial No. 389,697, filed August 4, 1989.

High gradient magnetic separation (HGMS) is

Another useful technique for performing

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typically accomplished by using a device having a separation chamber in which a wad of magnetic stainless steel wire is disposed between the poles of a conventional electro- or superconducting magnet and serves to generate large field gradients around the wire which exert a strong attractive force on target substance-bearing magnetic particles.

A commercially available high gradient magnetic separator of the type described immediately above is the MACS device made by Miltenyi Biotec GmbH, Gladback, West Germany, which employs a column filled with a non-rigid steel wool matrix in cooperation with

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a permanent magnet. magnetic field gradient produced in the vicinity of the steel wool matrix attracts and retains the magnetic particles while the non-magnetic test medium passes through and is removed from the column. Similar magnetic separators employing a steel wool matrix for separating colloidal size magnetic components from a slurry containing same are also disclosed in U.S. Patent Nos. 3,567,026, 3,676,337 and 3,902,994. In the last mentioned patent, the separator is provided with a magnetic wool matrix capable of movement into and out of the influence of a magnetic field as a continuously moving element.

It has been found that the steel wool matrix of such prior art HGMS devices often gives rise to non- specific entrapment of biological entities, other than the target substance, which cannot be removed completely without extensive washing and resuspension of the particles bearing the target substance. Mo;eover, the size of the column in many of the prior art HGMS devices requires substantial quantities of experimental materials, which limits their use in performing various important laboratory-scale separations. In addition, the steel wool matrix may be harmful to certain sensitive cell types.

identified above is the subject of co-pending and commonly owned U . S . Patent Application Serial No. 588,662, filed September 26, 1990. The separator of this co-pending application comprises magnetic means featuring a pair of confronting magnets external to the container and a magnetic gradient intensifying means positioned within a container holding the test medium.

In operation, the enhanced

A useful magnetic separator that avoids problems

The magnetic particles adhere to the magnetic means within the container which serves to separate or

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are of relatively simple construction and operation, relying only on gradient intensifying means external to the separation chamber, and yet maximizing magnetic field gradients, and which reduce entrapment of non-

35 target substances, eliminate loss of immobilized

remove the particles from the test medium. U.S. Patent No. 4 , 6 6 3 , 0 2 9 relates to an HGMS

device which is stated to be an improvement with respect to devices employing a magnetic wool matrix as

5 the magnetic field gradient intensifier, as well as to devices relying on differences in magnetic susceptibility of particles in a fluid to effect

6 3 separation. The I 029 patent describes an apparatus for continuous magnetic separation of particles from a slurry according,to their magnetic moment, by passing the slurry through a separator comprising a non- magnetic canister with a magnetized wire or rod extending adjacent to the canister. The wire is magnetized by a magnetic field to create a magnetization component transverse to the longitudinal axis of the wire, thereby to provide a field gradient extending everywhere within the canister space and exerting a radial force on particles passing through the canister. Depending upon the orientation of the

20 magnetic field relative to the canister, diamagnetic particles in the slurry can be attracted toward the wire and paramagnetic particles repelled, or vice versa, for a magnetic field usually rotated by 90°

with respect to the plane of the canister. 25 From the foregoing review of the prior art, it is

apparent that HGMS affords certain advantages in performing medical or biological analyses based on biospecific affinity reactions involving colloidal magnetic particles. Nevertheless, it would be

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30 desirable to provide HGMS apparatus and methods which

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target substance due to shear forc.es or collisions with other biological entities, and enable use of standard microtiter plate wells, and the like. Such a development would clearly be of practical utility in

particularly in immunoassays and cell sorting. 5 conducting various laboratory-scale separations,

SUMMARY OF THE INVENTION It is an object of this invention to provide

magnetic separation apparatus and methods capable of generating a high gradient magnetic field within a non-magnetic test medium to separate magnetically responsive colloidal particles from the test medium. Unlike relatively larger size magnetic particles which tend to settle out of an aqueous medium, magnetically responsive colloidal particles remain suspended in an aqueous medium for an indefinite period, thereby making them readily accessible to target substances.

at'least one container and magnetic means capable of

medium within the container. The container has a peripheral wall with an internal surface area and is adapted to receive the test medium with the magnetically responsive colloidal particles therein

2 5 (hereinafter Itthe test medium being separated"). As

will be described in further detail below, the magnetic field gradient generating means is disposed outside the container and provides an IIopenIl field gradient inside the container, wherein the magnetic

internal wall surface of the container than it is in the test medium most distant from the wall.

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The magnetic separator of the invention comprises

20 generating a high gradient magnetic field in the test

30 field is stronger in the test medium along the

If the test medium being separated is in a steady state, e.g., in a batch-type operation, suitable

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containers include microtiter wells, test tubes, capillary tubes closed at one end, or other non- magnetic cylindrical walled vessels defining a chamber for performing the desired separation. Furthermore, a plurality of test samples may be processed simultaneously_through the use of a carrier capable of holding more than one sample container. preferred form, the carrier includes means for holding a plurality of containers around the periphery of the carrier.

In a

If the test medium is to pass continuously through the separator, a suitable container is a conduit or tube having openings at each end. containers are preferably non-magnetic, e.g., glass or plastic, and of cylindrical configuration. Preferably, the container has an inlet opening at one end for receiving the test medium which is exposed to the high magnetic field gradient at the center portion of the container. In this particular embodiment, the container may also have one or more non-magnetic baffles spaced apart within the container between the two ends. The baffles are dimensioned to restrict the cross-sectional area of the passageway in which the test medium flows through the container to a region surrounding the axis. The baffles are preferably inclined radially downwardly along the direction of flow, to guide toward the wall any magnetically responsive colloidal particles coming into contact therewith. The conduit preferably has laterally spaced apart outlet means disposed at the end opposite the inlet. One outlet means may be provided along the periphery of the outlet end of the container for collecting the magnetic particles. Another outlet means is centrally positioned at the outlet end for discharging the test medium.

Such

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In a particularly preferred embodiment, the magnetic field generating means may comprise sets of four or six permanent magnets or electro-magnets. magnets are arranged so as to define a cavity which accommodates the container. In this embodiment, the polarity and pasitioning of the magnets located on the opposite sides of the cavity are such as to produce flux lines which generate a high gradient magnetic field within the test medium in the container. The magnets may be housed in a ferromagnetic yoke, preferably of cylindrical configuration, which serves to enhance the field strength produced by the apparatus. this llmultipolell arrangement is characterized by a very strong magnetic field near the edge of the cavity and by virtually no magnetic field at the center of the cavity. Accordingly, magnetic particles in the test medium adjacent the wall of the container near the edge of the cavity are subject to considerably greater magnetic force than particles in the test medium furthest from the wall of the container, toward the center of the cavity where the field strength falls away to zero.

The magnetic means may advantageously comprise magnetic flux concentrating means, particularly, if the cavity defined by the magnets is much larger than

The

The magnetic field gradient produced by

the cross-section of the container or carrier. Suitable for this purpose are pole pieces of various geometries, which are magnetized or magnetizable via an induced field. The magnets comprising the magnetic means may be attached magnetically to, or otherwise joined to the yoke, e.g. by cementing with epoxy, to hold the magnets in a fixed position relative to one another.

The physical properties of the magnetic particles

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preferably used in the practice of this invention, particularly the relatively small particle size, permit a level of operating efficiency which, insofar as is known, has not been achievable heretofore. Furthermore, by controlling the quantity of magnetic particles added to the test medium, relative to the exposed surface area of the wall of the container in contact with the test medium and controlling the orientation of such exposed surface, so as to be substantially coextensive with the flux lines of the magnetic field, it is possible to cause the magnetic particles to adhere along the exposed surface of the container wall in a substantially single layer, corresponding in thickness to about the size of the magnetic particles and any substance or material borne thereby. By operating in this way, occlusion of nonspecifically bound substances in the immobilized magnetic particles is virtually negligible.

particles from a non-magnetic test medium in accordance with the methods of the invention, the particles are initially dispersed in the non-magnetic test medium, forming a stable suspension therein. The magnetic particles typically comprise a receptor capable of specific binding to a target substance of interest in the test medium. separate target substances from test medium in a steady state, a suitable container holding the test medium and the receptor-magnetic particle conjugates are placed in the magnetic separator for batch-wise processing. The external magnetic means disposed around the container produces a magnetic field gradient in the test medium, which causes the magnetic particles to move toward the wall and to become adhered thereto.

In separating magnetically responsive colloidal

If it is desired to

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In the method of the invention which employs a plurality of containers held in a carrier, the magnetic field gradient causes the magnetically responsive colloidal particles in the test medium to move toward and adhere to the wall of each container closest to the-magnetic means. this method, the orientation of the wall of each container in the carrier relative to the magnetic means may be controllable.to cause the particles to adhere more uniformly around the wall of each container.

In accordance with

In another embodiment of the method of the invention, the test medium being separated may be flowed through the separator. The magnetic field gradient intensifying means produces an IIopenIl f i e l d gradient of sufficient strength to pull the magnetic particles from the test medium moving at a pre- determined rate and to adhere them to the wall. The non-magnetic test medium is discharged from the container at the outlet end. of this method, in which the container includes one or more baffles, the test medium to be separated is poured into the inlet opening at one end of the conduit. As the test medium moves through the conduit, the magnetic particles in the test medium are attracted by the magnetic means toward the wall of the conduit and thereby comes in contact with the baffles. The baffles are arranged to cause the particles to be carried toward the wall of the separation vessel. The magnetic means may be operable to cause the particles to become adhered to the interior wall of the separation vessel, or to permit particles to move down the wall for collection at one or more outlets

In a related embodiment

provided along the periphery of the wall opposite the inlet. The test medium may

at the end be removed at

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an outlet laterally spaced from the particle outlet(s) in the center portion of the conduit at the end opposite the inlet end.

In carrying out the methods of the invention, the non-magnetic test medium may be removed from the separator while the magnetic particles are retained on the walls of the container and subjected to further processing, as desired. involving biospecific affinity reactions in this way, resuspension of the magnetic particles bearing the target substance is effectively obviated. Accordingly, this method substantially reduces the processing time required for, and thus the cost of, bioanalytical testing.

the present invention, that in performing the separation methods described herein, there is no need to remove certain reagents present in excess in the test medium before addition of a magnetic common capture agent, such as colloidal magnetic goat anti- mouse Fc particles. practical advantage in that obviating removal of unbound labelling monoclonal antibody, for example, not only simplifies separation procedures generally, but particularly enhances cell viability in bioanalytical procedures performed on cells, e . g . ,

cell separations of clinical interest. In this connection, the present invention also provides a method for separating a membrane-containing bioentity, having a characteristic determinant, from a test sample by the steps of: introducing into the test sample a receptor having binding specificity for the

By performing analyses

It has also been discovered, in accordance with

This discovery may be used to

determinant, in an amount sufficient to bind the receptor to the determinant and to provide excess receptor in the test sample; introducing into the test

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sample, in the presence of the excess receptor, a multivalent capture agent which binds to the determinant-bound receptor, thereby to form a bioentity-receptor-capture agent complex, at least one of the determinant-bound receptor and the capture agent having associated therewith colloidal magnetic particles; and separating the complex from the test sample under the influence of a high gradient magnetic field.

From the foregoing summary, it will be appreciated that the present invention provides separation apparatus and methods of relatively simply construction and operation which enable the efficient and effective separation of target substance-bearing magnetic particles from a test medium.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a perspective view of a magnetic

separation apparatus embodying the present invention, portions of the apparatus being broken away for the purposes of illustration;

Figure 1;

of the invention having a magnetic means comprising six magnets;

Figure 4 is a fragmentary perspective view of a

related embodiment of the invention in which several containers are held in a carrier;

Figure 5 is a view in perspective of another embodiment of the invention in which a flow-through test container is provided with internal baffles;

Figure 6 is a vertical section through the apparatus of Fig. 5 .

Like characters of reference designate like parts

Figure 2 is a plan view of the apparatus shown in

Figure 3 is a plan view of a related embodiment

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in those figures of the drawings in which they occur.

DESCRIPTION OF PREFERRED EMBODIMENTS

and methods will now be described in detail with reference to the drawings.

the present invention have particular utility in various laboratory and clinical procedures involving biospecific affinity reactions. In such procedures, particles are used which are at once magnetically responsive and colloidal (i.e., particles which are superparamagnetic and capable of remaining in suspension in a non-magnetic test medium), and which comprise a receptor capable of binding the substance of interest in the test sample. In the present method, after the receptor binds the target substance, the magnetic separator removes the magnetic particles from the test medium via HGMS.

Such biospecific affinity reactions may be

Preferred embodiments of the present invention

The magnetic separation apparatus and methods of

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employed in testing biological samples for the determination of a wide range of target substance, representative of which are cells, cell components, cell subpopulations (both eukaryotic and prokaryotic), bacteria, parasites, antigens, specific antibodies, specific biological factors, such as vitamins, viruses and specific nucleic acid sequences, as in the case of gene probe analysis. Thus, the magnetic separation apparatus and methods of the invention may be used to carry out cell separations for the analysis or isolation of cells including, by way of example: T- cells from a T-cell lymphoma cell line; B-cells from a B-cell lymphoma cell line; CD4 positive cells from leukocytes; lymphocytes from leukocytes; tumor cells from normal cells; and stem cells from bone marrow

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cells. The method

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of th invention may also be used for immunospecific isolation of monocytes, granulocytes and other cell types; removal of rare cells; depletion of natural killer cells; determination of reticulocytes;.and assays for neutrophil function, e.g., for determining changes in membrane potential, performing oxidative burst analysis, phagocytosis assays and opsonization studies.

apparatus and method may be used in bacterial or parasite separation or analysis, including the separation of various bacteria and parasites from fecal matter, urine, sludges, slurries and water (e.g., ground water or streams). The present invention may also be used in separating various bacteria in food products (liquids to solids) sputum and urine.

The preferred magnetic particles for use in

Similarly, the present magnetic separation

carrying out this invention are particles that behave as true colloids. Such particles are characterized by their sub-micron particle size, which is generally less than about 200 nanometers (nm.) (0.20 microns) and their stability to gravitational separation from solution for extended periods of time. materials are composed of a crystalline-core of superparamagnetic material surrounded by molecules which may be physically absorbed or covalently attached to the magnetic core and which confer stabilizing colloidal properties. The size of the colloidal particles is sufficiently small that they do not contain a complete magnetic domain, and their Brownian energy exceeds their magnetic moment. As a consequence, North Pole, South Pole alignment and subsequent mutual attraction/repulsion of these

Suitable

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colloidal magnetic particles does not appear to occur even in moderately strong magnetic fields, which contributes to their solution stability. Accordingly, colloidal magnetic particles are not readily separable from solution as such even with powerful electromagnets,-.but instead require a relatively high gradient magnetic field to be generated within the test medium in which the particles are suspended in order to achieve separation of the discrete particles.

properties can be prepared as described in U . S . Patent No. 4 , 7 9 5 , 6 9 8 , the entire disclosure of which is incorporated by reference in the present specification, as if set forth herein in full.

typically prepared from appropriately prepared body fluids, such as blood, urine, sputum or secretions. It is preferable to add the colloidal magnetic particles to the test medium in a buffer solution. A suctable buffer solution for this purpose comprises a mixture of 5% bovine serum albumin ("BSA") and 95% of a biocompatible phosphate salt solution, optionally including relatively minor amounts of dextrose, sodium chloride and potassium chloride. The buffer solution should be isotonic, with a pH about 7. The protein serves to decrease interactions which tend to interfere with the analysis. The target substance may be added to the test medium before, after or simultaneously with introduction of the magnetic particles. The methods of the invention take advantage of the diffusion controlled solution kinetics of the colloid magnetic particles, which may be further enhanced by the addition of heat to the test medium. The test medium is usually incubated to promote binding between the receptor and any ligand of

Magnetic particles having the above-described

For cell separations, the test medium is

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interest present therein. conducted at room temperature or at a temperature slightly above the freezing point of the test medium (i.e., 4OC). Occasionally, incubation can be carried out at 37OC. The period of incubation is normally of relatively short duration (i.e., about 2-15 minutes). The test medium may be agitated or stirred during the incubation period to facilitate contact between the receptor and ligand.

If a small percentage of the buffer solution is replaced by a suitable anionic polyelectrolyte, binding of the receptor to a material other than the target substance in the test medium (i.e., non- specific binding) is noticeably reduced. Satisfactory results have been obtained using a commercial scale inhibitor sold under the name Tamol 850, which is available from Rohm and Haas, Philadelphia, PA. Tamol 850 is sold as an aqueous solution of polymethacrylic acid, having a molecular weight of 12,000 (weight average), total solids of 29-31%, density of 9.9 lbs./gal. (at 25OC), a Brookfield viscosity of 125-325 (at 25O) and a spindle/speed of #2 @60. The addition of about 0.1% to about 3% Tamol 850 (on an active basis) to the phosphate buffer will generally be adequate to reduce non-specific binding in the practice of the invention.

In carrrying out the method of the invention in which various bioentities are magnetically separated from a test medium containing excess receptor, antibody which binds selectively to the bioentity of interest is generally used as the receptor. Monoclonal antibody is preferably used for this purpose. However, non-antibody receptors, including antigen for antibody-producing cells or antigen processing cells, lectins, such as concanavilin A,

Incubation is typically

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20

soybean agglutinin, wheat germ agglutinin and the like, biotin-labeled reagents or hapten-labeled reagents, may be used, if desired.

binding selectively to the receptor, so as to form a bioentity-receptor-capture - agent complex. Suitable capture agents include Protein A or Protein G, where Ig is used as the receptor; avidin, where a biotin- labeled reagent is used as the receptor; and anti- hapten, where a hapten-labeled reagent is used as the receptor.

The capture agent is a substance capable of 5

Either biotin or a hapten may be used to

15

20

25

30

35

"2. /

facilitate capture of lectin receptors, e.g., concanavilin A, soybean, agglutinin or wheat germ agglutinin, which bind selectively to membrane- containing bio-entities comprising carbohydrate or glycoprotein components. Hapten/anti-hapten pairs which are suitable for this purpose include dinitrophenol (DNP)/anti-DNP, fluorescein/anti- fluorescein or arsanilic acid/anti-assanilic acid. The' capture agent preferably comprises colloidal magnetic particles which enable separation via high gradient magnetic field; however, the receptor may comprise colloidal magnetic particles, if desired.

interest is allowed to occur, magnetic separation of the colloidal magnetic particles from the test medium

After binding of the receptor to the substance of

is performed using the apparatus and methods of the

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t'r

present invention. The test medium is placed in or passed through a suitable separation vessel for batch- wise or continuous processing, as desired. Magnetic .

means disposed around the external periphery of the container generate a high gradient magnetic field or flux within the test medium transverse to the walls of the container. The magnetic means comprises a plurality of north magnetic poles and a plurality of

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21

south magnetic poles positioned alternatively around the container. In accordance with certain preferred embodiments of the invention, four or six magnets surround the container with equidistant intervals

substantially uniform along the walls of the container. The high gradient generated in this way causes the magnetic particles to migrate toward the surface of the wall of the container to which the particles adhere, rendering them easily separable from the test medium.

magnetic separator in accordance with the present invention. having a peripheral wall 2 5 and an open top for receiving the test medium. also includes four magnets 27 which define a cavity 33 adapted to receive the container. The magnets 27 are equally spaced along the inner surface of cylindrical

mounted, e.g., by cementing. The magnets may, if desired, be provided with magnetic flux concentrating means, such as pole pieces 29, which permits variation in the lateral dimension of cavity 3 3 to accommodate

which make the field more uniformly radial. As shown in Figures 1 and 2, the container used

to hold the test medium is a microtiter well. The container is positioned substantially coaxially with

position, the magnetic field in the test medium 35 adjacent to the wall approaches the magnetic field generated by the magnets. In contrast, there is virtually no magnetic field in the part of the

5 therebetween, so that the magnetic field is

10

Figures 1 and 2 illustrate an embodiment of a

The separator 21 comprises a container 23 15

The magnetic separator

20 feZromagnetic housing or yoke 31, on which they are

25 containers of different dimensions, or pole pieces

30 cavity 3 3 defined by the magnet faces. In that

35 container located along the axis 37 of cavity 33,

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i.e., the test medium most distant from the wall.

test medium provides ah ample surface area for adherence of the colloidal magnetic particles. advantage of the magnetic separator of the invention, when utilized under the conditions described above, is that by appropriately regulating the quantity of magnetic colloid, the particles tend to deposit substantially uniformly upon surfaces in contact with the medium where the magnetic gradient is high. As a result, particles may be caused to be deposited on a broad portion of the internal surface area of the wall in what is effectively a single layer, as opposed to multiple layers or particle agglomerates, which tend to entrap potentially interfering substances, as when formed on a smaller surface, such as occurs in magnetic separators of the prior art. In experiments employing IgG-bearing magnetic colloid, for example, it was found that when colloid mixed with enzyme was collected on the side of a separation vessel, enzyme became trapped as the colloid began to accumulate in layers. In the practice of this invention, by contrast, the colloidal magnetic particles are sufficiently thinly deposited on the container surface that there is virtually no entrapment of potentially interfering substances. To this end, it is preferable that the portion of the container wall surface in

The wall of the microtiter well exposed to the

An

contact with the test medium be selected so that the wall's aggregate collecting surface area is greater, by a factor of about 2 , than the surface area that wold be occupied by all of the magnetic particles in the test medium, if deposited in a substantially continuous single layer.

For permanent magnet devices, the magnetic field strength of the external magnetic means (magnets 27 in

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2 5

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23

Fig. 2) at the pole faces should be in a range of ---- 4-10 KGauss, and more preferably between about 6-8 KGauss. The preferred distance between each-magnet and the container shown in Figures 1 and 2 is generally about 0.1 cm. to about 2.0 cm. with the most preferable distance being about 0 .5 cm. the external magnet or magnets should be great enough, and the distance between the magnets and the container 23 for the test medium short enough to give efficient separation of the magnetic particles. magnets, considerably higher field strengths at the pole faces, on the order of 15-30 KGauss, can be achieved. In this way, very high gradient fields are obtained.

The cavity 3 3 should be formed by the magnets with sufficient excess space for manipulation of the container 23. For example, an elevating mechanism (now shown) may be positioned in the cavity to raise and support the container 23 in the position ilhstrated.

The pole pieces 29 illustrated in Figures 1 and 2 may be fabricated of any magnetizable material. pole pieces are shown in the form of magnetized bodies of trapezoidal cross-section, but may be fabricated in other shapes, such as triangular cross-section. These configurations aid in concentrating the .magnetic flux emanating from magnets 27. The pole pieces 29 may be held in place on the faces of magnets 27 by magnetic attraction. Alternatively, magnets 2 7 may be fabricated such that the pole piece is unitary with the body of the magnet.

Figure 3 illustrates another embodiment of the magnetic separator of the invention that is similar to the magnetic separator shown in Figures 1 and 2. The separator 121 shown in Figure 3 comprises a container

The field strength of

With electro-

The

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123 in the form of a cylindrical tube or the like. The container may be closed at one end, e.g., a test tube for batch-wise processing, or open at both ends, e.g., a capillary or larger diameter tube for continuous processing. and an opening-for receiving the test medium being separated. means represented by six magnets 127 which are equally spaced around the container.

separator of the invention illustrated in Figures 1 and 2. In Figure 4, the separator 221 comprises a plurality of containers 223 held by a carrier 241 and a magnetic means external to the carrier. container has a peripheral wall 225 and an open top. The magnetic means comprises four magnets 227 having curved faces disposed concentrically around the

The container has a wall 125

The separator also comprises magnetic

Figure 4 illustrates a variation of the magnetic

Each

j e n e d ~ p l a i A r carrier. The carrier 241 comprises a,non-magnetic base

243’,-.with separate fitted into the dimensioned to hold securely one of the containers 223 in an upright position.

the magnetic separator of the invention. separator 321 comprises a cylindrical container 323 with openings at either end to permit the test medium being separated to flow through the container. -The separator also comprises four magnets 327 which are spaced around the container with substantially equal intervals therebetween.

Figures 5 and 6 illustrate another embodiment of The magnetic

The container illustrated in Figures 5 and 6 has a peripheral wall 325 that defines an inner cross- sectional space 362 inside the container. One end of the container has an inlet port 363 that is

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dimensioned to permit entry of test medium to the central portion 365 of the inner cross-sectional space between the wall and the central portion. The baffles are also inclined downwardly along the direction of flow toward wall 325. Hence, in passing through the container, the-magnetically responsive colloidal particles are attracted by the magnets 327 onto the baffles and toward the wall. The baffles guide the particles along to a point at which the field gradient becomes sufficiently strong to cause the particles to adhere to the wall. two Outlets. One outlet 369 is along the periphery of the wall for collection of the particles. outlet 371 is aligned with the axis of the container for discharge of the non-magnetic test medium.

The container also has at least

A second

The magnetic particles may be separated from the test medium using the magnetic separator apparatus described above, after the test medium has been subjected to the magnetic field for a sufficient time to'cause the magnetic particles to migrate and adhere to the container wall. The non-magnetic components of the test media may be removed by decantation or aspiration, with the container still in the separator. A buffer, a wash liquid, or the like may then be added, while the container remains within the separator, to contact the wall for washing the adhering magnetic particles substantially free of any residual non-magnetic components. If desired, the wash liquid may be removed and the process repeated. If it is advantageous to resuspend the magnetic particles, the container may be removed from the magnetic field and manipulated to allow the magnetic particles to be dislodged from the walls and resuspended in a suitable liquid medium, e.g., to facilitate analysis.

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26

The magnetic separation apparatus and methods of the invention permit advantageous use of diffusion controlled solution kinetics of the primary incubation mixture. Moreover, various analytical procedures, including quantitative determinations, may be performed on the magnetically immobilized colloid. Such steps include washes for removal of nonspecifically bound substances, secondary immunochemical reactions or detection reactions (e.g., enzymatic, fluorescent or chemiluminescent reactions).

The ability to retain the magnetic particles adhered to the wall of the container after the test medium has been removed is of considerable utility. Certain operations are more efficiently carried out in this way, such as washing or rinsing the target substance, e.g., cells or labeled components of a reaction mixture, while avoiding a separate resuspension step. In addition, secondary reactions such as those involving the interaction of labeled immunoreactive agents with a target substance carried by the magnetic particles may be performed more efficiently with the particles adhered to the wall. Here again, resuspension of the colloidal magnetic particles is avoidable. Furthermore, in performing enzyme-labelled immunoassays in accordance with the present invention, substrate incubation-is preferably carried out directly on the colloidal magnetic particles immobilized in the separation apparatus.

Performing the magnetic separation method of the invention batchwise, i.e. in a steady-state system, as described above, instead of in a flow through system, has certain advantages. Immobilized magnetic particles bearing the target substances are not dislodged due to collisions with other particles. Moreover, batchwise operation eliminates dislodgment

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of immobilized magnetic particles due to shear forces produced by a flowing test medium. the adherence of the magnetic particles to the wall is sufficiently strong to permit washing, secondary reactions, and interactions with other reagents to occur without appreciable dislodgment of the magnetic particles from the wall. of the magnetic particles to the container wall is maintained to some extent even if the container is removed from the magnetic field before further reaction with, or treatment of the particles.

desirable to be able to employ a magnetic separator adapted for use in a flow through process. example, flow through processes are very desirable for separating large quantities of material of a uniform type. Surprisingly, the separator of the invention avoids the problem of dislodged particles noted above that commonly occurrs when employing a flow through process.

In general, the magnetic particles are relatively easily separated from the container wall after removal from the magnetic field. The particles may be dislodged by contacting the wall with a modified buffer solution, or a bath sonicator. Alternatively, the particles may be collected as they are dislodged from the wall with a probe sonicator.

those described above in which near neighbor pole faces are of opposite polarity create high gradient fields wherein the magnitude of the gradient is limited only by the field density at the pole face and the distance separating opposing poles. Compared with gradient fields produced by inducing magnetic fields on ferromagnetic material (fine wires, rods, spheres

In other words,

In addition, the adherence

Nevertheless, in certain applications it is very

For

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Multipole magnetic separation devices such as

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etc.) placed in external fields, the multipole devices of the invention have substantial advantage because the field strengths at the center of the opposing poles is zero. limited by the ability to produce fields greater than zero. -.

Hence, the gradients produced are only

The following examples further describe in some detail the manner and process of making and using the present invention and set forth the best mode contemplated by the inventors for carrying out the invention, but are not to be construed as limiting the invention. All temperatures given in the examples are in O C , unless otherwise indicated.

EXAMPLE 1 To demonstrate the utility of high gradient

fields produced by multipole apparatus in effecting magnetic separation in accordance with the present invention, quadrupole and hexapole devices were constructed and tested. permanent rare earth magnets were used (Crumax 355, Crucible Magnetics, Elizabethtown, KY); however, electromagnets could also be used, if desired. Quadrupole and hexapole devices similar to those shown in Figs. 1-3 were made comprising a yoke in the form of a 5 cm. long cylindrical steel pipe of 2 . 5 inch internal diameter (obtainable from plumbing supply distributors) and bar magnets disposed on the inner walls of the cylinder. Crumax 355 bar magnets 1.27 x 1.27 cm. by 5 cm. (magnetized through one of the 1.27 cm. dimensions) were mounted on the inner surfaces of the cylinder, with the long dimension of the bar magnet parallel to the axis of the cylinder. For the

For this demonstration,

quadrupole apparatus, four bar magnets were employed, placed at 9 0 ° , 180°, 2 7 0 ° and 360° with respect to a

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10

15

30

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29

plane normal to the axis of the cylinder. hexapole apparatus was similarly constructed except that six bar magnets were placed at 6 0 ° , 120°, 180°,

240°, 300° and 360° around a cylindrical yoke. In both the quadrupole and hexapole apparatus, the polarity of the magnets alternated around the yoke. For the quadrupole and hexapole apparatus thus constructed, opposing magnet faces were 3.4 cm. apart (pole face gap radius=1.7 cm) and the field density at each pole face measured 5 . 5 KGauss using a Gauss meter (Applied Magnetics Laboratory, Baltimore, MD). As the field at the center was 0 the average field gradient was 3.2 KGauss/cm.

To evaluate the effectiveness of the quadrupole and hexapole apparatus for radially separating small magnetic particles or cells to which magnetic particles are bound, the following experiments were done. A bovine serum albumin coated magnetic colloidal (60 nm. diameter particles) was prepared by the method described in Example 1 of Owen et al., U . S .

Patent No. 4,795,698. A small agglomerate (200-300 nm) of this magnetic colloid was formed by the addition of NaCl which causes a partial salting-out of the colloid, as described in copending U.S. Patent Application Serial No. 389,697, filed August 4, 1989. Such agglomerates have magnetic separation properties similar to cells to which are bound magnetic colloids bearing specific cell surface antibodies. Hence, the preparation was useful for assessing how cells would separate in a multipole high gradient field apparatus. This agglomerate is also useful for evaluating separability of the colloid, but in a more reasonable period of time, as compared with separation of the

described agglomerate in single microtiter wells (0.7

The

actual colloid. Using 250 ul aliquots of the above- e

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cm. i.d.) the local magnetic fields within these multipole devices were determined by placement of the wells at different locations within the field and observing the collection of agglomerate on the wall of the microtiter well after clearing from the test medium (generally 3-5 minutes). From these experiments, it was found that for the quadrupole and hexapole apparatus the field forces are radial and substantially axisymmetric over a radius of about 60-

70% of the pole face gap radius. performed at greater radii showed that magnetic agglomerate was not collected on the outer radii of the microtiter wall in a uniform manner, whereas highly uniform radial collection was observed at the lesser radii.

To further substantiate the axisymmetry of collection within the radial regions described immediately above, an open-top cylindrical glass vessel of 2 cm. internal diameter containing the above described magnetic agglomerate was placed in the center of the quadrupole or hexapole devices and separation observed. dilutions of the agglomerate so that deposits of magnetic particles on the walls of the vessel could be observed at several densities. In all cases, the deposits on the walls were observed to be substantially uniform. in a vessel placed substantially off -center in the apparatus, non-uniformity of collection was observed on the cylinder wall closest to the pole faces.

would separate similarly, a dextran coated colloid ( 8 0

nm diameter), prepared as described in U.S. Patent 4 , 4 5 2 , 7 7 3 to Molday, was placed in the glass vessel described above and allowed to separate for 10

Separations

This was done at several

When separation was performed

To demonstrate that a non-agglomerated colloid

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10

15

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minutes, by which time a uniform deposit was obtained on the walls of the vessel. It is thus reasonable to expect that colloidal magnetic particles are rapidly separable in apparatus of the type described aboye, in which the gradient fields are higher than those generated -in the present experiment.

Although the magnetic field at the center of the apparatus was measured to be zero, magnetic particles were collected from the center of the vessel. Thus, it appears that the thermal energy of small magnetic particles in combination with mixing due to thermal influences in the solvent are sufficient to cause diffusion to the high gradient region of the vessel. Field uniformity and added axisymmetry can theoretically be achieved by appropriate curviture of the pole faces and magnets used to construct multipole devices. By minimizing the gap between near neighbor magnets further reduction in field irregularities near these regions can be achieved. .

EXAMPLE 2 Mammalian cells were separated using the

multipole apparatus of Example 1, above. The target substance in this experiment was human T-cells (ATCC Accession No. CCL119 CCRF-CEM) which were maintained in cell culture under conditions available from ATCC. Cells were either labeled with chromium 51 (CRS1) by the procedure of GGV Klaus, LvmDhocvtes, a Practical Amroach, IRL Press Ltd., p. 144 (1987), or they were fluorescently labeled with acridine orange according to a procedure well known in the art. After labeling by either procedure, cells were washed in the usual manner. Cells were resuspended to approximately 3 x l o 6 cells/ml in a cell-compatible isotonic phosphate

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10

15

20

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30

32

buffered saline solution containing 0.1% sodium azide, pH 7.2, supplemented with 1% (w/v) BSA. A dextran coated colloid (80 nm. diameter) which was made by the Molday procedure, as described in U.S. Patent No. 4,452,773, and showed very high non-specific binding to cells, was used to magnetically label cells. The final concentration of the colloid was 17 ug iron/ml. of cell suspension.

Fluorescently labeled cells were placed in open- top glass cylindrical vessels of 1.76, 2.38 and 3.17 cm. i.d., respectively. These vessels were placed in the center of the quadrupole or hexapole separation apparatus which were mounted on the stage of an inverted microscope (Zeiss Axiovert 35 M, Thornwood, NY) in such a way that separation in all parts of the vessel was observable via fluorescent microscopy of the cells. For the two smaller diameter radii vessels employed, magnetically induced migration of the cells to the peripheral wall of the vessel was completely uncform. When the 3.17 cm. diameter vessel was used, irregularities of collection were observed on the periphery at locations corresponding to the gaps between near neighbor magnets. Thus, for the

quadrupole apparatus there were four regions ( 9 0 ° , 180°, 270’ and 360O) where excessive collection of cells occurred; and with the hexapole six such regions were observed. Hence, by selecting a separation vessel of appropriate dimensions for a given separation apparatus, substantially uniform deposition of the magnetic particles on the walls of the vessel may be achieved.

To determine how to move radially from

EXAMPLE 3

efficiently cells can be induced the center of a multipole device

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(where the field is zero), radiodabeled cells were placed in a 2.38 cm. i.d. cylindrical vessel which was then placed at the center of the hexapole device constructed as previously described. test medium were carefully removed periodically from the center of the vessel and counted for CR", from which depletion of cells from this region was calculated. By three minutes, 30% of the cells were removed from the central region, by seven minutes 77% of the cells were removed and by twelve minutes 8 6 %

removal was observed. Thus, even in the case of cells which exhibit essentially no thermally induced motion there is sufficient mixing of cells such that they can be pulled from the very center of such devices. This experiment demonstrates that cells flowing through a cylindrically shaped container placed in a multipole apparatus are removable in an efficient manner. Using appropriate flow parameters and with appropriate

Aliquots of the

baffling a multipole device could be used for continuous removal of cells.

EXAMPLE 4

A quadrupole separation apparatus was constructed from a cylindrical steel yoke having 5.78 and 8.32 cm. inner and outer diameter, respectively, and measuring 5.10 centimeter high, and 4 Crumax 355 permanent bar magnets of dimensions 0 . 6 3 5 x 2.54 x 5.10 cm., magnetized through the 2 . 5 4 cm. dimension. The four magnets were placed on the inner surface of the cylinder at 90° , 180°, 270Oand 360Oand aligned such that the 5.10 cm. dimension of each bar magnet was parallel to the axis of the yoke and the 2 . 5 4 cm. dimension was radially aligned. As with the above described quadrupoles, opposing magnet faces were of

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the same polarity and near neighbor magnets had opposite polarities at their faces. bar magnets properly aligned, i.e., at 9O0to each other, arcuate spacer elements made of aluminum were machined and fitted into the spaces between bar magnets. This -arrangement created a quadrupole having a gap between opposing pole faces of,.70 cm. Gauss meter, the field at the pole face surface was measured at 5.95 KGauss and zero at the center. the average field gradient for this assembly was 17 KGauss/cm. To determine how well this separation apparatus removed colloidal sized magnetic particles from solution, a 6 cm. length of glass tubing, 0.30 cm. inner diameter, was inserted into the quadrupole along its axis. This tube was connected to a syrine pump (Harvard Apparatus, Dedham, MA) such that test medium could be introduced into the glass tubing positioned upright in the field of the quadrupole and subsequently removed for inspection. alhmin-coated colloidal magnetic particles of 8 0 nm. diameter, made according to Example 1 of Owen et al., U . S . Patent No. 4,795,698, and suspended in 20 mM

phosphate buffer, pH 7.5, were drawn up into the glass tubing, positioned in the magnetic field and separation was allowed to take place. suspensions of this colloidal material containing from 20-100 ug/ml of iron were subjected to the magnetic field gradient, complete separation occurred in about 6 minutes, as determined by spectrophotometric examination of the solution pumped out of the separation tube.

material on the inner surface of the separation tube,

separated, after which the mother liquor was pumped

To maintain the

Using a

Thus,

Bovine serum

When

TO determine the distribution of the colloidal

levels Of colloidal material similar to the above were

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from the tube using the syringe pump and the magnetically deposited material was allowed to dry on the inner surfaces of the tube. dried was thereafter not affected by magnetic

5 gradient. From inspection of these tubes, it was apparent that material - _ is evenly deposited on the inner surfaces of the separation tubes.

Deposited material so

15

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1.

EXAMPLE 5

Typically, when cells are immune specifically retrieved by means of labelling with cell-specific monoclonal antibody (MAb), it is customary to incubate cells with MAb, wash out excess MAb and next incubate with a multivalent common capture agent, e.g., second antibody or avidin on a suitable solid support, such as magnetic particles. The purpose of removing excess MAb is to prevent agglutination between non-cell bound MAb and the multivalent common capture agent, and also to preserve binding sites on the common capture agent. Toxdemonstrate the efficiency of external gradient field devices for performing immune specific cell separations, while at the same time obviating removal of excess MAb before the addition of common capture magnetic colloid, the following experiments were done. Affinity purified goat anti-mouse Fc (GAMFc) (Jackson Immunoresearch Lab, West Grove, PA) was coated along with bovine serum albumin (BSA) onto colloidal magnetic particles as described in U.S. Patent No. 4,795,698 for use as the common capture agent. The final concentration of GAMFc and BSA on the colloid were 0.7 mg and 0.05 mg, respectively, per milliliter of final colloid. CEM cells were Cr’’-labelled and resuspended to a final concentration of 2.5 x 106/mL

in PBS as described in Example 2 above. To 1.35 mL of

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cell suspension, 85 UL of anti-CD 4 MAb (Gen-Trac, Plymouth Meeting, PA) was added at a concentration of 0.1 mg/mL (total 8.5 micrograms MAb) with mixing and incubated five minutes. Next, 1.35 mL GAMFc magnetic colloid was added (67.5 micrograms Fe), mixed, and incubated for five minutes; 2.6 mL of this mixture was immediately placed in -a fabricated polystyrene separation vessel (internal dimensions 10 mm x 8 mm x 51 mm high). Finally, this separation vessel cell was placed in a quadrupole device.

The quadrupole was constructed from four bar magnets which were 5.0 cm long, 1.6 cm wide and 1.9 cm deep and they were magnetized through the 1.9 cm dimension. The flux density at the pole faces was 6.6 KGauss and the radius of the quadrupole was 1.1 cm. Thus, the gradient for the device so constructed was 6 KGauss/cm. The 10x51 mm face of the separator vessel was placed directly on one of the pole faces, hence separation took place radially through the 8 mm dimension of the vessel.

Magnetic separations were performed for five minutes, ten and fifteen minutes, respectively, on identically prepared samples. All operations were performed at room temperature.

5% BSA) also included 1% Tamol-850 (Rohm-Haas, Philadelphia, PA) to prevent non-specific binding of colloid to cells. Cell supernatant was removed from the separation vessel with a 20 gauge needle placed at the back of the vessel (nearest the center of the quadrupole) and aliquots counted for CrS1, and also visually counted on a hemocytometer. For these experiments, it was found that 4 8 , 83 and 90% of the cells were pulled to the surface of the vessel facing

Further, the GAMFc colloid buffer (PBS containing

36

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30

35

37

the pole piece in 5, 10 and 15 minutes, respectively. In the absence of MAb, or with non-specific MAb no depletion of cells took place. For experiments done

in which cells were washed free of excess MAb after MAb incubation, followed by addition of GAMFc colloid, cell depletions over the 5, 10 and 15 minute magnetic separation periods were considerably lower.

It is interesting to note that the amount of MAb left free in solution far exceeds that on cells. If it is assumed that the cell has 50,000 receptors and can bind that many MAbs, then less than 0.05 micrograms of MAb is consumed, or 8 . 4 5 micrograms remain in solution. solution and the fact that there is excess colloid in solution, separation is readily accomplished. At the dilution of GAMFc colloid used for cell separation, agglutination reactions between MAb remaining free in solution and excess magnetic capture agent are not visually apparent and, indeed, if small complexes do e x h , they are not deleterious to the separation.

removing excess MAb has very beneficial implications for cell analysis, in addition to simplifying procedures. cells be expeted by reducing manipulations, but obviating removal of excess MAb permits the use and exploitation of lower affinity antibodies which, when added in excess, can be "forcedu1 on to receptors, but which readily disassociate when excesses are removed. Additionally, this methodology enables the use of relatively low-affinity, non-antibody receptors including lectins, such as concanavilin A, soybean agglutinin or wheat germ agglutinin for cell labeling via selection of an appropriate common capture magnetic colloid.

Despite that quantity of MAb in

The ability to do such separations without

Not only would improved viability of

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From the foregoing experiments, it is clear that a multipole arrangement is useful for separating small magnetic particles from a fluid containing same, e.g., for analytical purposes. flow sy'stem of essentially the same design could be constructed for removing colloidal magnetic material from a moving carried fluid, where such capability is desired in a particular application, such as bioprocessing.

have been described and exemplified above in terms of certain preferred embodiments, various other embodiments may be apparent to those skilled in the art. For example, the method of the invention may be scaled up to accommodate large volumes of material for various industrial processing applications, especially bioprocessing. The invention is, therefore, not limited to the embodiments specifically described and exemplified, but is capable of variation and modification without departing from the spirit of the invention, the full scope of which is delineated by the appended claims.

Alternatively, a continuous 5

10 While various aspects of the present invention

15

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5. A magnetic separator as claimed in claim 4 ,

wherein said container is of cylindrical configuration and each of said magnets has a curved pole face, said faces being disposed in a circle substantially

I

the magnetic particles within the said magnetic particles toward

such particles to be adhered means comprising a plurality and a plurality of south

alternately around the

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yoke.

3 . A magnetic separator as claimed in claim 2, wherein said magnetic means generates a field of about 5 to about 3 0 KGauss.

4 . A magnetic separator as claimed in claim 1, wherein said magnetic means comprises multiple magnets.

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WHAT is CLAIMED IS: 1. A magneti

particles from a no magnetic particle comprising:

peripheral wall receiving the te

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concentric with said container.

6. A magnetic separator as claimed in claim 4 ,

wherein said magnetic means also comprises a magnetic flux concentrating means associated with at least one of said magnets.

- - . 7. A magnetic separator as claimed in claim 6,

wherein said flux concentrating means is a pole piece.

8 . ' A magnetic separator as claimed in claim 1, wherein said magnetic means comprises at least four magnets surrounding said container with poles of like polarity confronting one another.

9. A magnetic separator as claimed in claim 1, wherein said magnetic means comprises six magnets spaced substantially equidistantly around the periphery of said container.

ful for separating tic test medium in d, said separator

gnetic containers, 1 surface area for

comprising:

for said plurality eans for engaging er edge of said

to said carrier adient that is the test medium les toward said agnetic means,

carrier; and

'I !

/--. ..

.-I

.-.

thereby c sing such particles to be adhered to said q y surface sub/ ithout substantial entrapment of interferring

A magnetic separator as claimed in claim 10,

spaced around said carrier, each of said magnets having a face confronting the face of one other magnet, said confronting faces being substantially diametrically opposed and being of the same polarity, said containers being positioned between the confronting magnet faces.

11. wherein said magnetic means compriseeou lT% m gnets

12. A magnetic separator as claimed in claim 11, wherein said magnetic means also comprises a magnetic flux concentrating means associated with each of said four magnets.

13. A magnetic separator as claimed in claim 10, wherein said magnetic means comprises six magnets spaced around said carrier, each of said magnets having a face confronting the face of one other magnet, said confronting faces being substantially diametrically opposed and being of opposite polarity, said containers being positioned between said confronting magnets.

14. A magnetic separator as claimed in claim 13, wherein said magnetic means also comprises a magnetic flux concentrating means associated with each of said six magnets.

or separating est medium in id separator

non-magnetic container having a tional space

comprising:

i-/ / -0'

...

.--..

/,- \ /inside the container, an inlet port at dimensioned to cause the test medium to inner cross-sectional space; and an o container at the end opposite one e non-magnetic test medium; and

receiving the

b) magnetic means nal to said container ic field transverse to

the wall of said cont thereby to generate a

magnetic field is strong in the internal surface area of the wall test medium alon

cross-sectio

se such particles to be adhered to said magnetic means comprising a plurality of

positioned alternately around the periphery of container.

.. 16. The separator as claimed in claim 15 wherein the shape of said cross-sectional space is generally cylindrical.

17. The separator as claimed in claim 15 wherein said magnetic means comprises four magnets spaced around said container with each of a face confronting the face of being of the same polarity, said container' being positioned between said confronting faces.

18. A magneti claimed in claim 15, wherein includes at e spaced between said ends inside said co affle confining flow of test medium prim e central portion of the cross-sectiona positioned so as to

' \. .. .

. . -

19. In a magnetic s comprising a container having a peripheral wall open top, and magnetic means positioned outside ontainer, a method for

substance from a non- magnetic test medium, the comprising the steps of:

uantity of magnetic particles compr capable of binding specifically to tance with said test medium under con ding of said receptor to said target s in target substance- bearing magneti

said magnetic particles id container; and b) introducin id test medium containing

c) positioning container holding said test medium wi 1 wall of said container transverse to in order to generate in,said test medium a ma ic field gradient in which the magnetic field is onger in the test medium adjacent to said wall of container than in the test medium most distant from wall and is operative upon the magnetic p est medium to attract said magnetic wall and cause such particles to

of magnetic lative to the t medium and urface area to cause 11 surface

\. .. .

... 1

.. .

L

20. A method as claimed i claim 19 including the step of maintaining the magne ic particles adhered to the exposed wall surface o said container while removing the non-magnetic 1. medium from said container. I

21. A method a in claim 2 0 including the particles adhered to said residual test medium from

step of washing‘the exposed wall surf ace said magnetic parti rom said container.

22. A method in claim 21 including the step of introducin container a resuspension medium and subje container to conditions causing removal of sa etic particles from said exposed surface and re ension of said magnetic particles in said

23. A meth gnetic particles

into a container p and positioned of applying a pheral wall to

generate a magnetic fi gradient in said fluid, said uid adjacent to from the wall, ed”to adhere to a plurality of south magnetic

from a fluid containing e, said method comprising:

poles positioned a1 ately around the periphery of said container; and

aid container.

, wherein said lled, relative

.. . ?,,

\

to the surface area wall exposed to said test medium, and the orie f said exposed wall surface area is controlled to said magnetic means, so as to cause said m rticles to adhere to said exposed surf ace stantially single layer corresponding in 0 about the size of said magnetic particles.

25. A metho to claim 23, wherein said magnetic particl a transition metal oxide substantially s a biofunctional polymer having availabl n sites, in proportions rendering said

to claim 25, wherein said transition metal is magnetite and said

protein or a polymer capable of being coupled to a

to claim 23, which is per-f ormed batchwise.

to claim 23 wherein an id colloidal magnetic

ing the presence or and in a test medium igand, said 'method

edim with a quantity rising or adapted to ligand, and with a said ligand, .under

conditions causing ding of said receptors to said

nd bearing magnetic

comprising:

. ...

.- . .

\ ! I

particles; said test medium into a 1 wall and an open top and

positioned ajacent to magnetic means capable of nsverse to said peripheral ield gradient in said test ing stronger in said test than in said test medium

most distant from the 1 and operative upon said id particles to adhere to comprising a plurality of

plurality of south magnetic around the periphery of

said test medium from said ic particles remain adhered

agnetic particles while said said wall to remove unbound

north magnetic poles a

said container;

container while sai

particles remain adhere substances; and

activity of said enzyme easure of the presence or d test sample.

n claim 29, wherein said rst and second receptors ic interaction with said

antigen.

claim 30, wherein said

32. A method ording to claim 29, wherein said ontacting said enzyme ducing a detectable r relative intensity

/- ---? . ~ ...

of said color by to a standard.

33. A met ing to claim 3 2 , wherein id enzyme is performed with red to said container wall-

detecting the ac said magnetic pa

3 4 . A met to claim 2 9 , wherein said quantity of ma es is controlled, relative to the exposed f the wall of the container and the orien exposed surface area is controlled relative said magntic means, so as to cause said ma to adhere to said exposed wall surface in substantially single layer corresponding in t ss to about the size of said enzyme and ligand netic particles.

35. A method rding to claim 29, wherein determinatio nd comprises conducting

eactions or detection reactions i and, with said magnetic

container wall.

ing from a test sample a having a characteristic

id test sample a icity f o r said

determinant, in amount sufficient to bind said to provide excess

test sample, in the ultivalent capture determinant-bound -receptor-capture

agent complex, least one of said determinant-bound receptor and d capture agent having associated

.. . '\,

38. A method according antibody is introduced

I . "

to claim 36, wherein an into said test sample as the

.' .

39. A method ng to claim 38, wherein said antibody is a mono

4 0 . A metho laim 38, wherein the test sample comprises capture agent int

colloidal magnet d with anti-Fc.

41. A meth aim 4 0 , wherein said colloidal magnet ise magnetite having a particle size about 0.50 microns.

4 2 . A met aim 36, wherein the e agent is selected A or Protein G.

receptor compri from the group

m 36, wherein said reagent and said

m 36, wherein said reagent and said

bioentity.

\ I I

I

/'%.

capture agent comprises anti-hapten.

4 5 . A method to claim 3 6 , wherein said receptor comprises labelled lectin seh3Aed from the group con concanavilin AI soybean agglutinin or whea lutinin and said capture agent comprises avidin.

4 6 . A method to claim 36, wherein said receptor comprises labelled lectin selected from the group co concanavilin A, soybean agglutinin or whe 1utinj-n and said capture agent comprises anti-

4 7 . A method ac to claim 36, wherein the n said test sample is from amount of receptor pre

about 2 to about 5 said determinant.

4 8 . A meth ating a biological cell having a characteri determinant from a test medium, said method comp

o said medium a monoclonal antibody having specificity for said determinan ufficient to bind said monoclonal antibody said determinant and to provide

said test sample; o said test sample, in the onal antibody, colloidal g anti-Fc which bind minant-bound monoclonal

antibody, thereby form a cell-monoclonal antibody-

conjugate from said high gradient magnetic

field.

G T I

I' , \. /

.\ i I

I i i \

i

/"',

UTILITY, Original U . S . or PCT D/O NO Priority

DECLARATION, POWER OF ATTORNEY AND POWER TO INSPECT

As a below named inventor, I hereby declare: that my residence, post office address and citizenship are as stated below next to my

name; that I verily believe I am the original, first and sole inventor (if only one .name is

listed below) or an original, first and joint inventor (if plural inventors are named below) of the Invention entitled: the specification of which fcheck one(s) applicable) - was filed

and was amended by Amendment filed (if applicable); [or] . --- X is attached to this Declaration, Power of Attorney and Power to Inspect;

as"PCT International/U.S. Application No. -

that I have reviewedtand understand the contents of the above identified specification, including the claims, as amended by any amendment referred to above; and

that I acknowledge the duty to disclose information which is material to the examination of this application in accordance with Rule 56(a) POWER OF ATTORNEY: As inventor, I hereby appoint DANN, DORFMAN, HERRELL AND SKI-, P.C. of Philadelphia, PA, and the following individual(s) as my attorneys or agents with full power of substitution to prosecute this application and to transact all business in the Patent and Trademark Office connected therewith: Henry H. Skillman, Reg. No 17,352 and Patrick J. Hagan,

I hereby give DANN, DORFMAN, HERRELL AND SKILLMAN, P.C. of Philadelphia, PA or its duly accredited representatives power to inspect and obtain copies of the papers on file

SEND CORRESPONDENCE

[37 CFR §1.56(a)]. t -

$ Reg. NO. 27,643. 3+ \POWER TO I N S P a

relating to this ANN, DORFMAN, BERRELL AND SKILLMAW, P.C: DIRECT PHONE CALLS TO: 1310 The Fidelity Building

(215) 545-1700 T m l a d e l p h i e , PA 19 109 Telefax: (215) 545-2750

I , S . Broad Street

I hereby declare that all statements made herein of my own knowledge are true and that all statements made on information and belief are believed to be true; and further that these statements are made with the knowledge that willful false statements and the like so made are punishable by fine or imprisonment, or both, under Section 1001 of Title 18 of the United States C=ode and that such willful false statements may jeopardize the validity of the application or any patent issued thereon.

Citizenship United States of America Countrv

(IF ANY)

p'. Signature

s/aa/q/ V

sidence-Eastoh, PA - State or Country Citizenship - United States of America

. Countrv - a Post Office Address: Post Office Address: 58 Green Drive 4565 Penacook Avenue Churchville, P A 18966 Easton, PA 18042, city State or Country zip Code City State or Country , Zip Code

Date 9-22- 9 \ & A Residenc2 Philadelma, PA v '- state or Countrv

Citizenship British Post Office-Address:

A

FOURTH JOINT INVENTOR (IF ANY)

. Full Name First Middle Last

Signature Date Residence

City State or Country Cit h e n s h ip Post Office Address:

600 W. Harvey Street Philadelphia, PA 19144 City State or Country Zip Code City State or Country Zip Code

e i i

_-.., : .. _...-._ _.-

..A; -. ... -

FIG. 2

FIG. 1

FIG. 3

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._ . .

i , " i

\ I I

e ,

i

FI G.

(323 363 f325 (

FIG. 5 F16.6

... .

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. .

FIG. 1

FIG. 2

I21

FIG. 3

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241

FI G. 4

363 tZ5 f 323 (

36

FIG. 5 FIG.6

_.__-- I

I [..- -7

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UNlTED S1A-a DEPARTMENT OF COMMERCE Patent and Trademark Office Address : COMMISSIONER OF PATENTS AND TRADEMARKS 1 Wefihington, D.C. 20231

s i

I

Thls is a mrnrnunication from ha examiner In charge of your applicarion. COMMISSIONER OF PATENTS AND TRADEMARKS

I

0 This action is made final. i & This application has been examined BResponsive IO communication filed 33-z5"fl I

A shortened statutory period for response to this adon Is set to expire 3 Failure to respond within the period for response will cause the application to becomelabandoned. 35 U.S.C. 133

Part I

4onth(s), 0 days from the date of this letter.

, !

THE FOLLOWING ATTACHMENT(S) ARE PART OF THIS ACTION: ' 8 ,

1. @I ~ o t i a of References cited 9. Notice of Art Clted by Applicant, PTO-1449. 5. Information on How to Effect Drawlng Changes, PTO-1474.

2. 4. @ Notice of Informal Patent Application, Form PTO-152

6. n Notice re Patent Drawing, PTO-948.

'

Port II SUMMARY OF ACTION

I.,& Claims I - I S am pending in the application.

are withdrawn from conslderation. 01 the above, daims

2 . 0 Claims have been cancelled.

3 . 0 Claims are ,allowed.

4. &f Claims I- d are rejected.

5 . 0 Claims are objected to.

\ 6.D Claims are subject to restriction br election requirement.

7. This a&licati?n has been filed with informal drawings under 37 C.F.R. 1.85 which are acceptable for examination purposes.

8 . 0 Formal drawings are required in response to this office action.

9. The corrected or substitute drawings have been receiwd on

\

. Under 37 C.F.R. 1.84 these drawings are 0 acceptable; 0 not acceptable (see explanation or Notice re Patent Drawing, PTO-948).

examiner; disapproved by the examiner (see explanation): ..

It

10. The proposed additional or substitute sheet(8) of dk ings , filed on . has (have) been 0 eppkved by the

11. The proposed drawing correction, Rled

12. Acknowledgement is made of the claim for priority under U.S.C. 11Q. The certified copy has- D been received U not been received

13. Since this application epppears to be In condition for allowance except for formal matters, prosecution as to the merits Is closed in

14. Other

, has been approved; 0 dlsapproved (see explanation).

been R l e d In parent appllcatlon, serial no. ; rid on . __

accordance wlth the practice under Ex parte Ouayle, 1935 C.D. 11; 453 0.G. 213.

. .

. . .. . , ' I ' . ..

. .

. . I . . - - I . . ._.

I. .

' I

I

'y )--

S e r i a l . No. 6 7 3 4 7 0

Art Unit 1306

-.- ...

-2-

R e s t r i c t i . o n to one of t h e f o l l o w i n g i n v e n t i o n s i s r e q u i r e d

under 35 U . S . C . 5 121':

I . Cla ims 1-1.8, drawn t o a magne t i c apparati-1s , c l a s s i f i e d

in C l a s s 21.0, suhc3.ass 222.

11. Claims 1 9 - 4 8 , drawn t o a magne t i c s e p a r a t i o n a s s a y ,

c l a s s i f i e d i n C l a s s 435 , suhcl .ass 7 . 2 1 .

Invent , ionf; I and I1 a r e r e l a t e d a s p r o c e s s and a p p a r a t u s f o r

i t s p r a c t i c e . The i n v e n t i . o n s a r e d i s t i n c t i f it c a n be shown

t h a t e i t h e r : (1) t h e p r o c e s s a s c l a imed c a n be prac t f iced by

a n o t h e r m a t e r i a l l y d i f f e r e n t apparatus or by hand, or ( 2 ) t he

a p p a r a t u s a s claj.med can be used t o p r a c t i c e a n o t h e r and

m a t e r i a l l y d i f f e r e n t p r o c e s s . (M.P.E.P. § 8 0 6 . 0 5 ( e ) 1 . In t1-ri.s

c a s e t h e process as clairned can be prac t i ced by another and

materially .dif f e r e i i t apparatus such a s with a niagiietic: microt i ter

p l a t e .

Because these i n v e n t i o n s a r e d i s t i n c t for t h e r e a s o n s g i v e n

a'bove and have a c q u i r e d a s e p a r a t e s t a t u s i n t h e a r t a s shown hy

t he i r d i f f e r e n t c l . a s s i f i c a t i o n , r e s t r i c t i o n for examina t ion

p u r p o s e s a s i n d i c a t e d i s g r o p e r .

During a t e l e p h o n e . c o n v e r s a t i o n wfith MI?. P a t r i c k .Hagan on

12-3.9-91. a g r o v i s i o n a l . e l e c t i o n was made with t r a v e r s e t o

p r o s e c u t e t h e i n v e n t i o n of I , c l a i m s 1-18. A f f i r m a t i o n of t h i s

e l - e c t i o n m u s t he made by appl . icai i t i n r e spond ing t o t h i s O f f i c e

action. Claims 19-48 a r e withdrawn from f u r t h e r c o n s i d e r a t i o n by

Se:cial. 'No. 67 467 8

A r k Unit 1.306

,.N'

.I._- / ,

.-_

- 3 -

t,he Examiner , 37 C . F . R . B ,1.142(h), a s b e i n g drawn t o a non-

e lected i n v e n t i o n .

Claims 1-18 a r e r e j e c t e d under 35 U.S.C. 8 1 1 2 , second

p a r a g r a p h , a s being i n d e f i n i t e f o r f a i l i n g t o p a r t i c u l a r l y pofint

o u t and d i s t i n c t l y c l a i m t h e subject m a t t e r whi.ch appl.i.cant

r e g a r d s .as the i n v e n t i o n . .

O n l i n e 1 9 of c l a i m 1, " t h e p e r i p h e r y " l a c k s a n t e c e d e n c e . .

Concernj.ng c l a i m 2 , t he l o c a t i o n . of t h e yoke i n r e l a - t i o n t o

t h e c o n t a i n e r and t h e magnetic: p o l e s r ec i t ed on l i nes 16-1.9 of

c l a i m 1 i s u n c l e a r .

On 1:ine 1 0 of c l a i m 1 0 , it i s u n c e r t a i n by "means f o r

engag ing the c o n t a i n e r " a s t o whether the p l u r a l i t y of e - o n t a i n e r s

: r e c i t e d o'n l i n e 5 a r e a l l engaged , o r i f o n l y one o f t h e

c o n t a i . n e r s i.s engaged . On l i n e 1 0 , " t h e o u t e r edge" l a c k s

a n t e c e d e n c e . On l i n e s 8 - 1 1 , i t i s u n c l e a r a s t o what s t r u c t u r e a

"non-magnet ic c a r r i e r " comprises. O n 1.iiie 1 4 , "upo nmayne t i c "

shou1.d be changed t o --upon magnetic--.

On l i n e 3.0 of c l a i m 15, it is u n c l e a r a s to w h i c h end "one

end" compr i se s (e.g., t h e o n e end reci ted on l i n e 7 ? ) .. On l i n e

1 4 , i t i s u n c e r t a i n a s t o which w a l l " t h e wall" r e f e r s t o . On

l i n e 2 4 , "the p e r i p h e r y " l a c k s a n t e c e d e n c e .

On l i n e 4 o f cl.aiin 1 7 , i t i.s u n c e r t a i n a s t o whether "o€ one

other magnet" re fe rs t o one of the f o u r inagnets r e c i t e d on l i n e

.-

s i j

_.-

I .

I

' i i I

Ser.:ial N o . 674678

A r t U n i t 1 3 0 6

-4 -

2 , o r t o an a d d i t i o n a l magnet .

Concern ing c1.aim 1 8 , t h e c l a i m f a i l s t o r e c i t e a d e q u a t e

s t r u c t u r e c a p a b l e of pe r fo rming t h e f u n c t i o n s r ec i t ed or1 l i . n e s 3-

7 .

The f o l l o w i n g i s a q u o t a t i o n of t he a p p r o p r i a t e paragraphs of 35 U . S . C . § 1 0 2 t h a t form t h e b a s i s for t h e r e j e c t i o n s under t h i s s e c t i o n made i n t h i s O f f i c e a c t i o n :

A p e r s o n s h a l l be e n t i t l e d t o a p a t e n t u n l e s s -- ( b ) t h e i n v e n t i o n was p a t e n t e d o r d e s c r i b e d i n a p r i n t e d p u b l i c a t i o n i n t h i s o r a f o r e i g n c o u n t r y o r i n p u b l i c u s e o r on s a l e i n t h i s c o u n t r y , more than one y e a r p r i o r t o t h e d a t e a€ a p p l i c a t i o n for p a t e n t i n t h e U n i t e d S t a t e s .

C l a i m s 1, 4 , 5 , 8 , and 15-1.8 a r e r e j ec t ed under 35 U . S . C .

§ 102(b) a s b e i n g a n t i c i p a t e d by I n a h a .

With respect t o c l a i m 1, Inaha discloses a non-magnet ic

contafiner 1 0 h a v i n g a p e r i p h e r a l w a i l w i t h an i n t e r n a l s u r f a c e

a r e a , and'rnagnetic means 1 2 ( s e e FIG.2) for g e n e r a t i n g a m a g n e t i c

f i e l d g r a d i e n t withi .n the c o n t a i n e r , t h e m a g n e t i c means

c o m p r i s i n g a p l u r a l i t y of n o r t h and s o u t h magne t i c poles

p o s i t i o n e d a l1 :e rna te ly around t h e p e r i p h e r y o f t h e c o n t a i n e r .

Concern ing c l a i m 4 , Inaha d i s c l o s e s magne t i c means

c o m p r i s i n g mu1tipl.e magnets 1 2 .

Concern ing claim 5 , Inaha d i s c l o s e s a c o n t a i n e r .1-0 of

c y l i n d r i c a l c o n f i g u r a t i o n , each magnet h a v i n g a cu rved po1.e face

( see FIG.2) , t h e p o l e f a c e s b e i n g d i s p o s e d i n a ci:rc:Le

s u b s t a n t i a l l y c o n c e n t r i c with t h e c o n t a i n e r .

4-e -..,g.. .

Regarding claim 8 , Inaha discl.oses a t l e a s t four magnets

‘ I

S e r i a l N o . 674678 -5-

A r t U J l i L 1306

surroi~ii id i iig the cqn t a i n e r w i t l i pol. e s of 1. i. k e pol. a r fi .I,y c!on f ron t :i.iig

one a n o t h e r ( s e e FIG.2).

W i t h respect ,to c l a i m 1 5 , Inaba d.i.scloses a non-niagnetj-c

c o n t a i n e r 1 0 hav ing a p e r i p h e r a l w a l l forming an j.iiner cross-

s e c t i o n a l space i n s i d e t h e c o n t a i n e r , an i n h e r e n t i n l e t p o r t 40

at; one end , and an i n h e r e n t o u t l e t port. 2 8 a t an end opposite

s a i d one end , magnetric means 3.2 e x t e r n a l t o t h e coril-.ai.iier fo r

a p p l y i n g a magne t i c fie1.d t r a n s v e r s e t o the w a l l o f t h e

c o n t a i n e r , t h e magne t i c rneans compri-sing a plurality of n o r t h a n d

s o u t h magne t i c poles p o s i t i o n e d a 1 t e r n a t e l . y a round t h e per:i.phery

of t he c o n t a i n e r .

Concern ing c l a i m 1 6 , I n a h a discloses a c o n t a i n e r 1 0 hav ing a.

cross sect:i.onal s p a c e t h a t is c y l i n d r i c a l .

Rega?d.ing c l a i m 3.7, Inaha discloses f o u r magnets 1.2 spaced

around t h e c o n t a i n e r w i t h the magnets h a v i n g a f a c e c o n f r o n t i n g

t h e € a c e of o n e other magnet , the f a c e s b e i n g of t h e same

pol.airi t y , the con ta i -ne r b e i n g p o s i t i o n e d between the c o n € r o n t i n g

€aces.

Regard ing claim 1 8 , Inaba discloses a t l e a s t one haE€le 16.

Claim 1 0 i s r e j ec t ed unde r 35 U . S . C . § 102(b) as b e i n g

a n t i c i p a t e d hy Wang.

With respect t o claim 10, Wang discloses a p l . u r a l i t y o € tion-

magne t i c c o n t a i n e r s 252-257 ( s e e F I G . 1) , a non-magnetic c a r r i e r

s e r i a l N o . 674678

A r t U n i t :1.306

.-- .

-6-

50 ( see FIG.2 aiid l i n e s 55-62 of cs l .3) hav ing nieans 52a for

engagi-ng l,he c o n t a i n e r a d j a c e n t t o an o u t e r edge o f the carrier,

magnetic means 20 e x t e r n a l ,to t h e c a r r i e r for g e n e r a t i . n y a

niayne t i c f i e l d g r a d i e n t i n each c o n t a i n e r .

/ T h e fol.I.owiny i s a q u o t a t i o n of 35 U . S . C . § 1-03 which forms the h a s i s €or a l l o b v i o u s n e s s r e j e c t i o n s s e t f o r t h j.n thyis Of€i.ce a c t i o n :

A p a t e n t inay n o t be o b t a i n e d though t h e i n v e n t i o n i s n o t i den , t : i , ca l ly d i s c l o s e d or descr i .hed a s s e t f o r t h in section 1 0 2 of this t i t l e , i f t h e d i f f e r e n c e s between t h e s u b j e c t m a t t e r sought t o be p a t e n t e d and t h e prior a r t a r e such t h a t t h e subject matter a s a whole would have been obvi.ous a t t h e time the j -nvent ion was made .to a p e r s o n hav ing o r d i n a r y s k i l l i n t h e a r t 1x1 which s a i d s u b j e c t matter p e r t a i n s . P a t e n t a b i l i t y s h a l l riot be n e g a t i v e d hy t h e manner j..n whj.cli the i n v e n t i o n w a s made.

S u b j e c t m a t t e r developed hy a n o t h e r p e r s o n which q u a l i f j - e s a s p r i o r a r t o n l y unde r s u b s e c t i o n ( f ) o r (g) of s e c t i o n 1.02 of t h i s t i t l e , s h a l l n o t p r e c l u d e p a t e n t a b i l i t y uncler t h i s sec t ' iqn where t h e s u b j e c t m a t t e r and t h e c l a jmed i n v e n t i o n were, a t the t i m e t he invent j .on was made, owned by the same p e r s o n or subject t o a n ob2i.gation of a s s ignmen t t o the same p e r s o n .

Cla ims 2 , 3 , 6 , 7 , and 9 a r e re jected tinder 35 U.S.C. § 103

as b e i n g u n p a t e n t a b l e o v e r Inaba i n view of L,ohmann.

Concern ing claims 2 , 6 , and 7 , Inaba cli.scloses t h e out'er

po3.e~ of t h e magnets a s being mounted on a cy l in ' d r i ca l . meta1l.j.c

s t r u c t u r e ( see FlG.2) , but f a i l s to speci.fy t h e whether t h e

s t r u c t u r e c o n s t i t u t e s a f e r r o m a g n e t i c yoke o r f l u x c o n c e n t r a t i n g

,<'e- .~ --&

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means /pole piece a s s o c i a t e d wi th a t ] . eas t one of t h e magnets .

Lohmann discloses an aizal.ogous s e p a r a t o r which i n c l u d e s mayne t i c

p01.e~ mounted to a f e r r o m a g n e t i c yoke 20/f lux c o n c e n t r a t i n g

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SerI.al No. 674678

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A r t - U n i t 1306

means/pole p i e c e a s s o c i a t e d w i t h a t l e a s t one of t h e magnets 30

( see FTG.2 and l i n e s 6-11 of c01 .2 ) I and t e a c h e s t h a t s u c h a

s t r u c t u r e comple t e s t he .magnet ic c i r c u i t s between t h e s t a c k s of

magnets t h e r e b y d i r e c t i . n y t h e l i n e s of magne t i c € lux toward t h e

c o n t a i n e r 6 j.n a more e f f i c i e n t and c o n c e n t r a t e d manner. I t would

have been obvi.ous t o have niodif ied t h e a p p a r a t u s o f Inaba so a s

t o have ii-~cl.i~cled .the f e r r o m a g n e t i c yoke a s suggested by TJolimann

i n order t.o i n c r e a s e t h e e f f i c i e n c y of t h e s e p a r a t o r .

Concern ing c1.ai.m 3 I t h e s e l e c t i o n o f magne t i c means c a p a b l e

of yenei.-ating 5 t o about, 30 R G a u s s wou1.d have been obv ious i.12

order t o o p t i m i z e t h e per formance o'f t h e a p p a r a t u s f o r the

des: i red flow r a t e , o r t y p e h a t e r i a l s b e i n g s e p a r a t e d .

Regardj-ng cl.aim 9 , Inaha f a i l s t o disclose six magnets

spaced s u b s t a n t i a l l y e q u i d i s t a n t l y a round t h e p e r i p h e r y of t h e

c o n t a i n e r , however, t h e s e l e c t i o n of such a number would have

heen obv ious i n o r d e r t o o p t i m i z e t h e magne t i c f i e l d j . n t e i i s i t y

f o r a g i v e n cross s e c t i o n a l a rea of t h e c o n t a i n e r , f low r a t e o€

m a t e r i a l , and t h e type of m a t e r i a l s b e i n g s e p a r a t e d , a s s u g g e s t e d

b y Lohmann who d i s c l o s e s the use of s i x magnets. .

Cla ims 1 1 - 1 4 wou1.d be a l l o w a b l e i f r e w r i t t e n t o overcome t h e

r e j e c t i o n unde r '35 1J .S .C. 5 11.2 and t o i n c l u d e a l l of t h e

limitations of t he base c l a i m and any i n t e r v e n i n g claims.

U . S . P a t e n t s 4209394, 3985649, and 564858 discloses v a r i o u s

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inagiletic s e p a r a t o r s .

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6- 0. %vu4-J MAlTHEW 0. SAVAGE

EXAM I MER ART UNIT 1306

M. Savage ( 7 0 3 ) 308-3854 F e b r u a r y 2 4 , 1 9 9 2

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U S DEPARTMENT OF COMMERCE Patent and Trademark Office

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Pro FORM 948 (HEV. 5-00)

THE PTO DRAFTSMEN REVIEW ALL ORIGINALLY FILED DRAWINGS REGARDLESS OF WHETHER THEY WERE DESIGNATED AS JNFORMAL OR FORMAL.

The drawings filed c

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, Patent )nd.Trademark..Office : . . . Address: ' COMMISSIONER OF' PATENTS AND';TRADEMARKS :

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' ' , ,..NOTICE OF: INFORMAL ~PPLICATION (Attachment to.Office ,d/ction).

.This application does. not conform with the -rules .governing 'applications for .the reasonb) . checked .below. The period within which t6' correct these requirements and avoid abandonment '

is set in the accompanying Office action;

A. A .new' oath or :declaration,.identifying this application,by the serial numbm .ind filing date is . ' ' ' . .

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1; 0 was not execuG&in accordrince with either 37 CFR 1.66 or 1.68. : 2. 0 .does not identify the city and stab2 or foreign country of residence of each inventor.

.4: 0 'does not state:whether the inventor is a sole or joint inventor. 5. 0 . .does not state that the person making.the oath ar declaration:.

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a. 0 has reviewed and understands the contents of the specification, including the claims, as amended by any amendment specifically referred to in the oath or declaration.

b. 0 believes the named inventor or inventors to be the original and first inventor or 'inventors of the subject matter which is claimed and for which a patent is sought.

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TO SEPARATE, ~pc-l> TOP AND BOTTOM' EDGES, SNAP-APART ANO, P--{CARD CARBON

FORM PTO-892 (REV. 3-78)

U.S. DEPARTMENT OF COMMERCE PATENT A N D TRADEMARK OFFICE

NOTICE OF REFERENCES CITED

I DOCUMENT NO.

J.S. PAT

SERIAL hRoUPART ATTACHMENT TO

NUMBER PAPEh 2 m I

APPLICANTIS)

UT DOCUMENTS I I SUB- IFILING DATE IF I CLASS I CLASS I APPROPRIATE NAME

I 1 FOREIGN PATENT DOCUMENTS

* A copy of this reference is not being furnished with this office action. (See Manual of Patent Examining Procedure, section 707.05 (a).)

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\3 In

THE UNITED STATES PATENT AND TRADEMARK

: Group Art

Examiner: M.O.

-. . .

re the Application

PAUL A. LIBERTI et a1

Serial No. 07/674,678

Filed: March 25, 1991

For: "Apparatus and Methods for Magnetic Separation Featuring External Magnetic : Me an s GRcyJP IB0

- _ CERTIFICATE OF MAILING UNDER 37 C.F.R. §1.8(a)

I hereby certify that this Correspondence is being deposited on April 30, 1992 with the United States Postal Service as first-class mail in an envelope properly addressed to COMMISSIONER OF PATENTS AND TRADEMARKS, Washington, DC 20231 I

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3 0 A f C i I 149> ?& s*/& u fl. Date of Certificate PATRICK J. HAGAN - _

Attorney for Applicant(s) 1.2

PTO Registration No. 27,943., - .

Hon. Commissioner of Patents and Trademarks

Washington, DC 20231

INFORMATION DISCLOSURE STATEMENT -1-

'In accordance with the duty of disclosure set forth in 37 C.F.R. 51.56, Applicants hereby submit the attached PTO Form 1449, listing references which the Patent Examiner is requested to consider and make of record in the above-identified application. Copies of the listed references are enclosed herewith.

References Cited in the Present Application

The article edited by Hunter et al. referenced on page 3 of the specification describes the use of magnetic particles to facilitate bound/free separations, particularly in immune and other bio-specific affinity reactions. The article discloses a magnetic separator in which tubes in a rack are positioned adjacent to a permanent magnet.

U . S . Patent Nos. 3,970,518 to Giaever; 4,018,886 to Giaever; 4,230,685 to Senyei et al.; 4,267,234 to Rembaum; 4,452,773 to Molday; 4,554,088 to Whitehead et al.; and 4,659,678 to Forrest et al. set forth on pages 3 and 4 of the specification provide examples of magnetic separation of small magnetic particles by external localized magnetic fields in analyses involving biospecific affinity reactions. The means of magnetic separation, if any, that is disclosed in the foregoing patents is based upon a local external magnetic - _ force.

The Robinson et al. article referenced on page 4 of the specification discloses that relatively large crystals of ferromagnetic materials retain permanent magnet characteristics after exposure to a magnetic field and tend to aggregate thereafter.

U . S . Patent No. 4,795,698 to Owen et al. referenced on pages 4 and 5 of the specification describes various biological applications of polymer-coated, sub-micron size magnetic particles that behave as true colloids.

The Dynal MPC-1 manufactured by DYNAL, Inc. Great Neck; NY and the BioMag Separator, manufactured by Advanced Magnetics, Inc. (AMI), Cambridge MA, both referenced on page 5 of the specification are commercially available magnetic separation units. These apparatuses are used to remove micron sized ferromagnetic particles from solution. AMI also offers two-piece magnetic separators consisting of a test tube holder which is designed to engage a magnetic separation rack containing permanent magnets. Also referenced on page 5 of the specification are similar magnetic separator, manufactured by Ciba-Corning Medical Diagnostics, Wampole MA which is . -provided with rows of bar magnets arranged in parallel and located at the base of the rack. These devices accommodate a plurality of test tubes in the test tube holder, with the closed end of each tube fitting into a

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recess between two magnetic separator

of the bar magnets in the rack. A

of generally similar design is offered by Serono Diagnostics, Norwell, MA.

Another commercially available magnetic separator of the type referenced above and on pages 6 and 7 of the specification is the MACS device made by Miltenyi Biotec GmbH, Gladbaeh, Germany. filled with a non-rigid steel wool matrix in cooperation with a permanent_magne,t comprising two pole pieces spaced to allow the column to fit between the pole pieces. operation, the enhanced magnetic field gradient produced in the vicinity of the steel wool matrix attracts and retains the magnetic particles while the non-magnetic test medium passes through and is removed from the column.

The MACS device employs a column

In

Copies of literature describing the above-referenced magnetic separators are enclosed.

The R.R. Oder article, C. Owen et al. reference, and the 1985 article of Kemshead et al. set forth on page 6 of the specification disclose that colloidal magnetic materials are not readily separable from solution as such, even'with powerful electro-magnets but, instead require high gradient field separation techniques.

The 1975 article of Hersh et al. referenced at page 5

--of the specification describes a specific application of a device for employing a single relatively inexpensive permanent magnet located external to a 'container holding the test medium. The device is used in performing magnetic solid-phase radio immunoassay.

,

U.S. Patent No. 4,141,687 to Forrest et al. also referenced at page 5 of the specification.describes an automated continuous-flow radioimmunoassay' system employing cellulose-coated magnetic particles. The automated system exemplified in the '687 patent includes

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elaborate electromagnetic traps which are operable in a pre-determined sequence by a programmer device under the control of a sample detector. individual pairs of facing magnets positioned external to and along a conduit through which flows the sample being tested.

The traps comprise

U . S . Patent Nos. 3,567,026, 3,676,337 and 3,9,02,994 collectively describe magnetic separators employing a steel wool matrix for separating colloidal size magnetic components from a slurry containing same. mentioned patent, the separator is provided with a magnetic wool matrix capable of movement into and out of the influence of a magnetic field as a continuously moving element and with a pair of magnets placed external to and on opposite sides of the matrix.

In the last

>

U . S . Patent No. 4,663,029 is stated to be an improvement with respect to devices employing a magnetic wool matrix as the magnetic field gradient intensifier, as well as to devices relying on differences in magnetic susceptibility of particles in a fluid to effect separation. The '029 patent describes an apparatus for continuous iiagnetic separation of particles from a slurry according to their magnetic moment, by passing the slurry through a separator comprising a non-magnetic canister with a magnetized wire or rod extending adjacent to the canister. The wire is magnetized by a magnetic field to create a magnetization component transverse to the longitudinal axis of the wire, thereby to.provide a field gradient extending everywhere within the canister space and exerting a radial force on particles passing through the canister. Depending upon the orientation of the magnetic field relative to the canister, diamagnetic particles in the slurry can be attracted toward the wire and paramagnetic particles repelled, or vice versa, for a magnetic field usually rotated by 90° with respect to the plane of the canister. The patent also discloses the use

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of two magnets positioned on opposite sides of the canister.

Although in the opinion of the undersigned, the foregoing references are the most pertinent of which t he undersigned is aware, no representation is made or intended that more pertinent references do not exist.

It is believed that the subject matter claimed in the present application constitutes a significant advance in the art, as represented by the references discussed above, and is deserving of patent protection.

Early and favorable consideration leading to prompt issuance of this application is earnestly solicited.

Respectfully submitted,

DANN, DORFMAN, HERRELL AND SKILLMAN A Professional Corporation Attorney for Applicant(s)

BY PATRICK J. HAGAN PTO Registration No. 27,643

Telephone (215) 563-4100 Facsimile (215) 563-4044

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9 STATES PATENT AND TRADEMARK OFFICE

1 1

Paul A. Liberti et al. 1 1

Serial No. 674,678 ) 1

Filed: March 25, 1991 1 1

For: APPARATUS AND METHODS ) 2 -

FOR MAGNETIC SEPARATION 1

MEANS 1

Examiner: M. S a v a g &/2*- e Group Art Unit: 1306

G & @ / Q ~ Response to Paper No. 2

Amendment A - _ * I

I

I r - L > > . FEATURING EXTERNAL MAGNETIC )

- .. -. -.- - 1 I' Certificate of Mailinq Under 37 CFR P1.8(a)

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I hereby certify that this correspondence is being deposited on June 5, 1992 with the United States Postal Service as<-first class mail in an envelope addressed to COMMISSIONER OF PATENTS AND TRADEMARKS, Washington, D.C. 20231.

' KJ-JLlW 1992- Date of Certificate Patrick J. Hagan

Attorney for Applicant PTO Reg. No. 27,643

AMENDMENT AND REQUEST FOR RECONSIDERATION UNDER 37 C.F.R. S1.111

In response to the Official Action dated March 5, 1992, please amend the above-identified patent application as follows :

Amend the claims as set forth in the attached Schedule A.

REMARKS The March 5, '1992 Official Action-and the references

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cited therein have been carefully considered and in view of the amendments presented herewith and the following: remarks, favorable reconsideration and allowance of this application are respectfully requested,

At the outset, it is noted that the restriction requirement previously communicated to the undersigned attorney by phone has been officially made of record. This requirement is respectfully traversed. It is respectfully submitted that the requirement for restriction is improper for

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- several reasons and should either be substantially modified, or withdrawn entirely.

The restriction requirement is premised on the unsupported assertion that the magnetic separation methods, as

claimed in claims 19-48, can be practiced by another and materially different apparatus than the apparatus claimed in claims 1-18. The Examiner has evidently overlooked the fact that independent method claim 23 and independent method claim 29 both specifically call for a magnetic separation apparatus having the characteristics of the apparatus of claim 1. That being the case, it is manifestly erroneous for the Examiner to conclude that the methods as claimed, at least with respect to claims 23-28 and 29-35, can be practiced by another and materially different apparatus.

With respect to the remaining method claims, namely, claims 19-22 and 36-48, it is respectfully submitted that these methods, which are practiced using apparatus of claims 1-18, are part and parcel of a single inventive concept, the objective of which is to enable the efficient and effective separation of magnetically responsive colloidal particles from a non-magnetic test medium by means of a high-gradient magnetic field generated within the test medium. The present invention allows magnetic separations to be carried out in such a way that the colloidal magnetic particles are caused to accumulate on the interior surface of the separation chamber in a substantially single layer, so as to minimize occlusion of undesired substances. In addition, when using the apparatus and method of the invention, there is no need to remove certain reagents, e . g . , unbound labelling monoclonal antibody, before introduction of a magnetic capture agent into the test medium. Thus, notwithstanding the Examiner's contention that the process as claimed can be practiced by another and materially different apparatus, it is submitted that the only practical way in which to perform the methods

claimed in claims 19-48 is by means of the apparatus of claims 1-18.

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Clearly the subject matter of claims 19-48 is directly related to the subject matter of claims 1-18. Consequently, the Examiner's search with respect to the apparatus of claims 1-18 would of necessity cover the same art areas as would a search of method claims 19-48. inclusion in the present application of method claims 19-48 would thus not materially affect the Examiner's workload, it is respectfully requested that the restriction requirement be withdrawn.

requested to note in any future Official Actions in this application the pending status of claims 19-48, until such times as claims 19-48, or any of them, are formally cancelled. The Form PTOL-326 which accompanied the March 5, 1992 Official Action in this case does not reflect the pending status of claims 19-48.

Since the

Also in this regard, the Examiner is respectfully

In order to be fully responsive, applicants hereby affirm the earlier provisional election of apparatus claims 1- 18.

Applicants hereby reserve the right to file one or more continuing applications, as provided under 35 U . S . C .

§120, on the subject matter of the non-elected claims. In the March 5, 1992 Official Action, claims 1-18

stand rejected under 35 U.S.C. S112, second paragraph, as allegedly indefinite. In this regard, the Examiner sets forth specific objections concerning claim terminology appearing in claims 1, 2, 10, 15, 17 and 18 at pages 3-4 of the March 5, 1992 Official Action.

Claims 1, 4, 5, 8 and 15-18 also stand rejected under 35 U.S.C. S102(b) as allegedly anticipated by the disclosure of Inaba, U.S. Patent 4,498,987, specific reference being made to Figure 2 of the Inaba patent.

Furthermore, the Examiner contends that claim 10 is anticipated by the disclosure of Wang, U . S . Patent 4,895,650, specific reference being made to Figure 1 of the Wang patent.

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Claims 2, 3, 6, 7 and 9 are further alleged to be unpatentable under 35 U.S.C. §lo3 based on the combined disclosures of Inaba, noted above, and Loman, U . S . Patent ~

3,402,820. According to the Examiner, it would have been obvious to modify the apparatus of Inaba by incorporating features of the Loman device, including magnetic poles mounted to a ferromagnetic yoke, flux concentrating means and pole pieces associated with at least one of the magnets, the purpose of such modifications being to complete the magnetic circuits between the stacks of magnets, thereby directing the lines of magnetic flux toward the container in a more efficient and concentrated manner. The Examiner further maintains that the selection of magnetic means capable of generating 5 to about 30 KGauss would have been obvious in order to optimize performance of the apparatus and that the selection of six (6) magnets disposed around the periphery of the container would have been obvious in order to optimize the magnetic field intensity for a given cross-sectional area of the container, flow rate of test medium and the type of material being separated therefrom.

, Also in the March 5, 1992 Official Action, the Examiner indicated that claims 11-14 would be allowable if rewritten in independent form to include all of the limitations of the base claim and any intervening claims, provided the claims, as rewritten, avoid the above-noted rejection under 35 U.S.C. S112, second paragraph.

(Eddelman) and 5,648,858 (Whiteacre et al.) were also made of record in the March 5, 1992 Official Action, but were not applied in rejecting any of applicants' claims.

U . S . Patents 4,209,394 (Kelland), 3,985,649

In accordance with the present amendment, claims 1, 2, 10, 15, 17, and 18 have been amended so as to point out with greater particularity the subject matter of the present invention. It is believed that these amendments clearly distinguish the magnetic separation apparatus of claims 1-18 over the art of record.

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In particular, claim 1 has been amended to specify that the magnetic means defines a receptacle for the container which is removably mounted in such receptacle. this amendment to claim 1 is provided in the present specification at page 21, lines 16-18 and at page 25, lines

Support for

30-35.

Claim 2 has been amended by stating that the yoke is disposed around the- container and that the magnetic poles are intermediate the container and the yoke. Support for this amendment is provided in Figures 1-3 and the description relative thereto provided in the specification.

structural features of the carrier, including that the magnetic means surrounds the carrier. amendment is provided in Figure 4 and the description relative thereto provided in the present specification.

Claim 15 has been amended to correct a self-evident misstatement in the characterization of the outlet port, as the function of the outlet is to discharge, and not to receive, the non-magnetic test medium. Claim 15 has been furthecamended to include a recitation of means to remove the

Claim 10 has been amended to recite further

Support for this

magnetic field and the magnetic field gradient to permit discharge of the magnetic particles from the container by gravitational force. Support for this amendment is provided by Figures 3 and 5 and the description relative thereto provided at pages 23-25 of the present specification.

As a result of the claim amendments presented herewith, it is believed that the above-noted 'ground of rejection based on 35 U.S.C. $112, second paragraph; has been overcome. In particular, any indefiniteness in claims 1 or 15 resulting from use of the term "periphery" has been eliminated by deleting the term periphery from claims 1 and 15. In claim 2, the location of the yoke is specified relative to the container and the magnetic poles. The amendment to claim 10 clarifies the structure of the carrier, including that' the carrier has means for engaging a plurality of containers and

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provides clear antecedent basis for the expression '!outer edgegt, used with reference to the carrier. The amendment to claim 15 makes clear that the Itone end" referred to therein is the inlet end and that the wall referred to therein is the container wall. The amendment to claim 17 makes clear that the one other magnetic referred to therein is one of the four ( 4 ) recited magnets. Claim 18, as amended, recites sufficient structure for performing the recited function of the separation apparatus set forth therein.

1992 Official Action are inapplicable to the claims as presently amended and should be withdrawn.

The prior art rejections set forth in the March 5,

Rejections under 35 U . S . C . §102(b) are proper only I when the claimed subject matter is identically disclosed or I described in the prior art. In re Arklev, 172 U.S, .P.Q. 524

(CCPA 1972). Applying this rule of law to the present case, the 35 U.S.C. §102(b) rejections of claims 1, 4, 5, 8 and 15- 18 based on Inaba, and of claim 10 based on Wang are clearly improper because the cited references plainly fail to disclose or describe the subject matter of the rejected claims.

._ There is no disclosure in the Inaba patent of a magnetic separation apparatus in which magnetic means define a receptacle for a container which is removably mounted in the receptacle, as now called for in claim 1. On the contrary, Inaba discloses an apparatus in which the container is provided with a screw conveyor and associated drive mechanism which is plainly not intended to be, nor is it, removable from the arrangement of magnetic plates provided thereon. This distinction cannot be viewed as trivial considering the function of the respective devices. Applicants' device, as described in the present specification, is intended for carrying out various laboratory and clinical procedures involving biospecific affinity reactions in which magnetic particles are separated from non-magnetic media, e.g., cell separations, immunoassays and the like. The container in applicants' apparatus must, of necessity, be removable from

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.- -

the magnetic.means in order to permit further processing of the magnetic particles, which typically occurs in carrying out such procedures. The device described in Inaba, on the other hand, is intended simply for the gross separation of magnetic particles from functional fluids of machine tools. magnetic particles are separated from the functional fluid by the joint action of the magnetic plates and the screw conveyor and are discharged -as waste, rather than undergoing further processing. Thus, there is no reason for the container component of the Inaba separation apparatus to be removably mounted in the magnetic plates and indeed, the separation device illustrated and described in the Inaba patent is not removed from the arrangement of magnetic plates provided thereon.

The

Amended claim 15 is also distinguishable from the disclosure of Inaba. Claim 15 now requires means to remove the magnetic field and the magnetic field gradient to permit discharge of magnetic particles from the container by gravitational force. No such means are provided in the apparatus of Inaba. The Inaba apparatus causes magnetic particle separation by means of a screw conveyor which acts in opposition to gravitational forces. Furthermore, those skilled in the art would not be motivated to eliminate the screw conveyor from the Inaba apparatus for the screw conveyor is essential to achieving the purpose of Inaba's magnetic separation apparatus.

Moreover, in view of the intended function of the Inaba apparatus, those skilled in the art would have no motivation or incentive for making modifications which would permit the container to be removably mounted in the magnetic means or to effect separation of magnetic particles by gravitational force.

claim 10, as now amended, is readily distinguishable from the.

magnetic separation rack disclosed in Wang. calls for magnetic separator having magnetic means surrounding

The magnetic separation apparatus called for in

Claim 10 now

... .

, i j I

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. a non-magnetic carrier, with the carrier having means for engaging containers adjacent to the outer edge thereof. The separation rack of Wang includes magnetic assemblies 2 4 , 2 6

and 2 8 which extend across the width of the base, but do not surround the containers supported by the rack. In view of this difference, Wang does not provide evidence of lack of novelty of the subject matter of claim 10.

tjl02(b) rejections of claims 1, 4 , 5, 8 , and 15-18 based on Inaba, and of claim 10 based on Wang are improper and should be withdrawn.

The 35 U . S . C . 5103 rejection of claims 2, 3, 6, 7

and 9 are also untenable in view of the present amendments. Claims 2, 3 , 6 , 7 and 9 depend, either directly or indirectly, from claim 1. As presently amended, claim 1 requires magnetic means defining a receptacle for a container, with the container being removably mounted in the receptacle, Inaba nor Lohrnan disclose or suggest such a structure. The structure of the magnetic separator disclosed in Lohman is substantially the same as that disclosed in Inaba and is intended for the same purpose. The Lohman separator, like that of Inaba, is not described as having a container removably mounted in a magnetic means serving as a receptacle and there is no reason for modifying the Lohman structure to make it correspond to the structure called for in applicants' claim 1. In short, the disclosure of Lohman does not make up for the manifest deficiency in the disclosure of Inaba, which have already been noted above. Thus, even if the disclosures of Inaba and Lohman are combined in the manner proposed by the Examiner, the resultant device does not provide response for all of the features recited in claims 2 , 3, 6, 7 and 9. That being the case, the 35 U.S.C. S103 rejection of claims 2, 3 ,

6 , 7 and 9 cannot stand. The Kellund, Eddelman and Whiteacre et al. patents

For all of the foregoing reasons, the 35 U . S . C .

Neither

were listed in the Notices of References Cited (Form PTOL-892) which accompanied the March 5, 1992 Official Action, but were

.I . .- . . -.

- 9 -

- not applied.against any of the claims. discussion of these three ( 3 ) references is required. it to say that none of these three ( 3 ) additional references, either individually or in any reasonable combination, provides evidence of unpatentability of the subject matter of claims 1-

In view of the amendments presented herewith and the

Therefore, no detailed Suffice

18.

foregoing remarks, it is respectfully urged that the rejections set forth in the March 5, 1992 Official Action be withdrawn and that this application be passed to issue and such action is earnestly solicited.


DANN, DORFMAN, HERRELL & SKILLMAN A Professional Corporation Attorneys for Applicant

BY ?&zAx&+&./

Patrick J. Hagan PTO Registration No. 27,643

Telephone: (215) 563-4100

Enclosure: Schedule A (Claim Amendments)

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_,,___._,.....,,.._, - .... . ) - . ... . . . . . .. . . . " .. , , , , .

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I,. /,-- /Serial No. 674,678 " / [ Filed: March 25, 1991

\.

-. -.- - - _ " -. . Examiner: M. Savage

Group Art Unit: 1306

I I.._

I.-- ---- SCHEDULE A '..+ </>L-

-----.-4_- _I -----,- __ CLAIM ._ , AMENDMENTS-------------~~ ____---*-

/ *_--- -- (,lac- 1. t(Amende3,) --I__ __- A magnetic separator for separating magnetic particles from a non-magnetic test medium in which said magnetic particles are suspended, the separator1 comprising :

wall with an internal surface area for receiving [the] said test medium; and

for generating a magnetic field gradient within said container, in which the magnetic field is stronger in the test medium along said internal surface area of [the] said wall than in [the] said test medium most distant from [the] said wall and is operative upon [the] said magnetic particles within [the] said test medium to attract said magnetic particles toward said surface area and cause such particles to be adhered to said area, said magnetic means comprising a

$'I a) a non-magnetic container having a peripheral

b) magnetic means [external to the container] I"

plurality of north magnetic poles and a plurality.of so th &h ex.\-c+iaf .dr&p magnetic poles positioned alternately around,,- of .

said peripheral wall [periphery] of said container and defininq a receptacle for said container, said container beinq removablv mounted in said receptacle.

../ -> (7

2. ;(Amended) ----- A magnetic separator as claimed in claim 1, wherein said magnetic poles are mounted on a cylindrical ferromagnetic yoke disposed around said container, said maqnetic poles beinq intermediate said container and said yoke.

*T

10. (Amended) A magnetic separator useful for separating magnetic particles from a non-magnetic test medium in which said particles are suspended, said 'separator comprising:

a) a plurality of non-magnetic containers,

Gt

. _----

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‘r I

I ’ , i‘ I I ’i j

..-~ I .

U . S . Serial No. 674,678 Filed: March 25, 1991 Page 2

having peripheral walls with internal surface area for receiving said test medium;

a b) a non-magne ic carrier for’said plurality ,A c/pdtt~ Q Lyse) 5qi) 5 4 3 ~ bein3 of containers, said 2arrier’- q enerallv planar and having

an outer edse with means for engaging said containers adjacent to said [the.] outer edge of said carrier; and

said carrier for generating a magnetic field gradient that is operative upon [upo n] magnetic test medium in each container to attract said particles toward said internal surface area closest to said magnetic means, thereby causing such particles to be adhered to said surface without substantial entrapment of interfering substances.

c) magnetic means surroundinq [external to]

particles within [the] said

- / Claim 11, line , after llcompriseslt insert --at

least--.

15. (Amended) A magnetic separator useful for separating magnetic particles from a non-magnetic test’medium in whi&h’said particles are suspended, said separator comprising:

peripheral wall forming4an inner cross-sectional space inside a) a non-magnetic container having a

avl rdcrvtd S O f f A C C ace+ and F

the container, an inlet port at one end dimensioned to cause the test medium to flow into the inner cross-sectional space, and an outlet port in said container at the.end opposite said one end for [receiving the] discharqinq said non-magnetic test medium; and

b) magnetic means external to said container and capable f applying a magnetic field transverse to [the]

magnetic field gradient in the test medium within said container, in which the magnetic field is [strong] stronser in [the] said test medium along said internal surface area of [the) said wall than in the test medium in a central portion

wall [of said container] thereby to generate a 9 said. ?d

I -- . .. . \

U . S . Serial No. 674,678

Page 3 ' Filed: March 25, 1991

of the cross-sectional space, and operative upon [the] said magnetic particles to attract said particles toward said surface area and cause such particles to be adhered to said area, said magnetic means comprising a plurality of north magnetic poles and a plurality of south magnetic poles

orv) s l c $ c r g d sdfquce 0-F- positioned alter-nately aGound [the peripheryln said peripheral B wall of said Fontainer: and

' discharqe of said maqnetic masnetic field q r a d i e n t . 4 particles from ''+''&=- said. bv qravitational force.

., /

c)!- said maqnetic field and said 9vl

2J ,9 Ir

Claim 17, line 4, after Itof onet1 insert --of the--; li / e 4, after llotherfll insert --of said--; / line 4 , change I1rnagnet1' to --magnets--.

~ ~~ -~ ~~

18. (Amended) A magnetic separator as claimed in claim 15, wherein said container includes at least one baffle spaced between said ends inside said container and transverse to said.- wall, said baffle havins an upstream surfack: a downstream surface and, a central openina to confine [said baffle confining] flow of test medium primarily through the central portion of [the] said cross-sectional space [area], [and being positioned so as] said upstream surface beins effective to guide magnetic particles contacting said baffle toward said wall.

Te.r p h c p d

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C L A I M S AFTER AMDT.

48

8

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CLAIMS NUMBER P A I D EXTRA FOR

- 48 3 0

- 8 = o

I N THE UNITED STATES PATENT AND TRADEMARK OFFICE

: Examiner: M. Savage

: Group A r t U n i t 1306

Paul A. L i b e r t i

For: APPARATUS AND METHODS FOR MAGNETIC SEPARATION FEATURING : EXTERNAL MAGNETIC MEANS

C e r t i f i c a t e o f Mail inq Under 37 CFR f1.8(a) 1 hereby c e r t i f y t ha t t h i s Correspondence i s being deposited on -June 5. 1992 wi th the United States Postal

Service as f i r s t - c l a s s mai l i n an envelope properly addressed t o COMMISSIONER OF PATENTS AND TRADEMARKS, Washington, DC 20231.

June 5, 1992 Date o f C e r t i f i c a t e PATRICK J. HAGAN

Attorney f o r Applicant(s) PTO Regis t ra t ion No. 27,643

Colrputation o f Additional Fee f o r Amw\hent

[ X 1 No Addi t ional Fee i s required. [

The fee has been ca lcu lated as shown.be1ow:

1 A check i s enclosed i n the amount o f S

FOR

EFFECTIVE TOTAL C L A I M S ' I

IND. .CLAIMS

I CLAIMS AS AMENDED

F I R S T PRESENTATION OF MULTIPLE DEPENDENT CLAIMS?

P E T I T I O N FEE FOR EXTENSION 1 month 1 I I I TOTAL I

--

*Applicant i s a Small 'Ent i ty , .as establ ished by a v e r i f i e d statement f i l e d In the event the check i s improper, or the fee ca l cu la t i on i s i n error , . the Commissioner is authorized t o charge

any underpayment o r c r e d i t any overpayment t o the account of the undersigned attoineys, Account No. 04-1406. A dupl icate copy o f t h i s sheet i s enclosed.

DANN, DORFMAN, HERRELL AND SKILLMAN A Professional CorDoration

Telephone: ( 2 1 5 ) 563-4100

BY PATRICK J. HAGAN PTO Regis t ra t ion No. 27,643

. _.-I-

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-- --. UNITED STATES DEPARTMENT OF COMMERCE Patent and Trademark Office

Address: COMMISSIONER OF PATENTS AND TRADEMARKS Washington, D.C. 20231

S E R I A L N U M B E R FILING D A T E F IRST N A M E D A P P L I C A N T A T T O R N E Y D O C K E T N O .

r EXAMINER 1

I - . .

~

P A P E R N U M B E R A R T UNIT

1 5- 1

DATE MAILED:

EXAMINER INTERVlEW SUMMARY RECORD

All participants (applicant, applicant's.representatiue, PTO personnel) :

d / n , (3)

(21 M'3fllro-a-e , (4)

Dam of interview '7- 3 jd 9

Type: @Telephonic 0 Personal (copy i s given to 0 applicant 0 applicant's representative).

Enhibit nhown or dwnonstration'conductad: 0 Yes H N o . I f yes, brief description:

Agt'eemsnt N w a s reached with respect t o some or all of the claims in question. 0 was not reached.

Claims discussed: 1 , 9'0 I , / 5. / 8 . d / 3 - 48 Identification of prior art discussed:

' .

Description of the general nature of what was agreed t o if an agreement was reached. or any other comments: 5 Q . & & d k d d shx*+,

(A fuller description, If necessary, and a copy of the amendments, if available, which the examiner agreed would render the claims rrllowable must be attached, Also, where no copy o f the amendments which would,render the claims allowable i s available, a summary thereof must be attached.)

Unless the paragraphs below have been checked t o indicate t o the contrary, A FORMAL WR tTTEN RESPONSE TO THE LAST OFFICE ACTION I S NOT WAIVED AND MUST INCLUDE THE SUBSTANCE OF THE INTERVIEW (e.g., items 1-7 on the reverse side of this form). I f a response to the last Office action has already been filed, then applicant is given one month from this interview date to providea Statement o f the substanceof the interview.

It i s not necessary for applicant to provide a separate record of the substance of the interview.

0 Since the examiner's interview summary above (including.any attachments) reflects a complete response to eech of the objections, rejections and requirements that may be present In the last Office action, and since the claims are now allowable. this completed form i s considered to fulfill the response requirements o f the last Office action.

PTOL-413 (REV. 1-841 Examiner's Signature

i-.,

I n t e r v i e w Summary, paper##5

Mr.l-lagan a g r e e d t o c h a n g e " t h e e x t e r i o r " t o --an e x t e r i o r

s u r f a c e - - on l i n e 18 o f claim 1 for p r o p e r a n t e c e d e n c e .

f i g r e e d t o amend claim lla t o s p e c i f y t h e b a s e o f t h e carr ier

as b e i n g g e n e r a l l y p l a n a r i n o r d e r t o c o r r e s p o n d w i t h t h e

s p e c i f i c a t i o n a n d FIG. 4 ( s e e e l e m e n t 243 i n FIG. 4). P r o v i d e d

a n t e c e d e n c e a n p a g e 24 o f t h e s p e c i f i c a t i o n for t h e 1 , i m i t a t i o n o f

" s a i d b a s e b e i n g g e n e r a l l y p l a n a r a n d h a v i n g a n o u t e r e d g e w i t h

means f o r e n g a g i n g s a i d c o n t a i n e r s a d j a c e n t t o 5 a i d o u t e r e d g e o f

s a i d car r ie r" a s r e c i t e d i n p a r a g r a p h b) o f claim 18, t h e

l i m i t a t i o n o f w h i c h h a d b a s i 5 i n FIG.4.

q y r e e d t o amend claim 15 at l i n e 6 t o p r o v i d e a n t e c e d e n c e ..

f a r t h e " i n t e r n a l s c u r f a c e area". Changed " c o n t a i n r r " t c r -- p e r i p h e r a l - - on l i n e 14 f o r p r o p e r a n t e c e d e n c e . Rmended l i n e 24

t o c l a r i f y w h i c h s i d e o f t h e w a l l t h e m a g n e t i c p o l e 5 w h e r e

l o c a t e d . Changed "means t o r e m o v e " w h i c h w a s c o n s i d e r e d new

matter t o - - s a i d c o n t a i n e r b e i n g r e m o v a b l y . . .mounted t o s a i d

m a g n e t i c means---- i n p a r a g r a p h c ) t o c o r r e s p o n d w i t h * the

s p e c i f i c a t i o n on l i n e s 38-35 o f p a g e 25. Flmended l i n e 2fi t o

c l a r i * F y w h i c h p a r t o f t h e c o n t a i n e r t h e p a r t i c l e s w e r e c o n t a i n e d .

a g r e e d t o c h a n g e " c o n t a i n e r " t o - - p e r i p h e r a l - - on l i n e 4 o f

claim 1 B for - p r u p e r a n t e c e d e n c e .

. __---

I

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_.-

Sev-ial No. 674676

Flrt U n i t 1386

...

fi M. Savage,’ 783-308-3854 July 3 1 g 199s

-3-

ngreed t o cancel non-elected claims 19-48 t o place t h e

application in condition f o r allowance.

See the attached examiners amendment.

ROBERT A. OAWSON SUPERVISORY PATENT EXAMINER

ART UNIT 136

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__..-...

.

SERIAL NUMBER 1 FILING DATE I FIRST NAMED APPLICANT ~ATTORNEY DOCKET NO.

Attachments: Examiner’s Amendment Examlnsr Intervlsw Summery Record. PTOL- 413

Nollce 01 References Clted. PTO-882 L( lnlormetlon Dlsclosure Cltellon. PTO-1448

- peasons for Allowance

IS I

NOTICE OF ALLOWABILITY

PART 1. I . This communication Is responsive to #t4 mdhd $ 6 - f - ’’ 2. @ Ail the claims belng allowable, PROSECUTION ON THE MERITS IS (OR REMAINS) CLOSED In thls application. If not included

herewith (or previously mailed), a-NoticB.01 Allowance And Issue Fee Due or other appropriate communication wlll be sent in due course.

3. @ The allowed claims are &fgfiJn? hU 5. 0 Acknowledgment is made of the clalm for priority under 35 U.S.C. 119. The certified copy has [-I been received. [-] not been

6. @ Note the attached Examiner’s Amendment. 7. d Note the attached Examiner Interview Summary Record, PTOL-413. 8. Note the attached Examiner’s Statement of Reasons lor Allowance. 9. 0 Note the attached NOTICE OF REFERENCES CITED, PTO-892.

10. fl Note the attached INFORMATION DISCLOSURE CITATION, PTO-1449.

I-lgi f a , & c $ i d are acceptable. 4. @ The drawings filed on 3 * 25- 4 71

received. 1-1 been filed in parent application Serial No. , filed on

PART I!. A SHORTENED STATUTORY PERIOD FOR RESPONSE to comply with the requlrements noted below is set l o EXPIRE THREE MONTHS FROM THE “DATE MAILED” indlcated on this form. Failure to tlmely comply wlll result In the ABANDONMENT 01 thls appllcatlon. Extenslons of time may be obtalned under the provislons of 37 CFR 1.136(a).

1. 0 Note the attached EXAMINER’S AMENDMENT or NOTICE OF INFORMAL APPLICATION, PTO-152, whlch discloses that the oalh or declaratlon IS deflcient. A SUBSTITUTE OATH OR DECLARATION IS REQUIRED.

2. APPLICANT MUST MAKE THE DRAWING CHANGES INDICATED BELOW IN THE MANNER SET FORTH ON THE REVERSE SIDE OF THIS PAPER.

a. 0

b. 0

c. 0

d. 0

Drawing Inlormallties are lndlcated on the NOTICE RE PATENT DRAWINGS, PTO-948, attached hereto or to Paper No. .,CORRECTION IS REQUIRED.

The proposed drawlng correctlon filed on has been approved by the examlner. CORRECTION IS REQUIRED. Approved drawlng correctlons are described by the examlner in the ettached EXAMINER‘S AMENDMENT. CORRECTION IS REQUIRED. Formal drawlngs are now REQUIREO.

Any response to this letter should Include In the upper right hand corner, the lollowlng inlormatlon from the NOTICE OF ALLOWANCE AND ISSUE FEE DUE: ISSUE BATCH NUMBER, DATE OF THE NOTICE OF ALLOWANCE, AND SERIAL NUMBER.

- Notlce 01 Informal Appllcatlon. PTO-152 - NOllce re Patent Drawlngs, PTO-948 - Llsling 01 Bonded Draftsmen - Other

PTOC-37 (REV. 2-85)

I USCOMM-OC 85-3744

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i

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S e r i a l No. 6574678

n r t U n i t 1386

... - .

Fln E x a m i n e r ' s Flmendment t o t h e r e c o r d a p p e a r s be low. S h o u l d

t h e c h a n g e s a n d / o r a d d i t i o n s be u n a c c e p t a b l e t o a p p l i c a n t , a n

amendment may be f i l e d ._ as p r o v i d e d by 37 C.F.R. § 1.312. T o

e n s u r e c a n s i d e r a t i o n o f s u c h a n amendment , i t MUST h e s u b m i t t e d

n o l a t e r t h a n t h e payment o f t h e I s s u e Fee .

W t h o r i z a t i o n f o r %I715 Examiner- ' s Rmendinent w a s g i v e n i n a

t e l e p h o n e i n t e r v i e w w i t h

On l i n e 18 o f claim 4

I a n e x t e r i o r surface--.

M r . Hagan on 7-31-92.

1, " t h e e x t e r - i o r " h a 5 b e e n c h a n g e d t o -- / /- J

J 0 / I On l i n e 9 a f claim 18, " b e i n g " h a 5 b e e n c h a n g e d t o --

/ i n c l u d i n g a ba5f3, s a i d b a s e be ing- - .

I/ a 0 '. / I n claim 15: o n l i n e 6, --an i n t e r n a l s u r f a c e area and-- h a s

0 0 / 0 / b e e n i n s e r t e d a f t e r i11=ormingp8 ; on l i n e 14, " c o n t a i n e r " h a 5 b e e n

J / / /

Cl7anQed t o - - p e r i p h e r a l - - ; on l i n e 24, -.ran e x t e r n a l 3 u r f a c e of - - - I 0 0

h a s b e e n i n s e r t 5 d b e f o r e "sa&"; on l i n e 26, "means t o remove" I

h a s b e e n c h a n q e d t o - sa id c o n t a i n e r b e i n g r e m o v a b l y moun ted t o

sa id m a g n e t i c means t o p e r m i t r e m o v a l a&-; on l i n e i2< "*bod B' I 0 I ' p e r m i t " 'has b e e n c h a n G d t o ---and--; an line i33, " c o n t a i n e r " has

I b e e n c h a n c e d t o - - . i n t e r n a l s u r f a c e area--.

/ On line (of claim 18, 1 * c d n t a i n S r l l I

p e r 1 ph e r a I -.- . / / Claims 19-48 h a v e b e e n c a n c e l e d .

h a 5 b e e n c h a n 6 d t o --

.

.. . \.,

i

/ / 1 J . tln page 24 o f ' t h e s p e c i f i c a t i o n : on l i n e 19, . -- .genEirally

/ /- / planar--- 13as 13e;en inssv-ter, i 1 3 e ~ o r e llnon-magne.t icll ; on 1 i n e d, 1 - 1

at a n o u t e r e d g e t h e r e o f - - - h a s b e e n i n s e r t e d a f e r " b a s e " . /

/ G /-.

a n o n - m a g n e t i c t e s t medi.um is disclosed hey-ein. The m a g n e t i c

!;epainak QP i n c l u d e s a non-magnet ic c o n t a i n e r - h a v i n g a p e r i p h e r a l

wall w i t h a n i n t e r n a l . 5uvface area for receiving t h e t e s t mertiuni,

and m a g n e t i c m e a n s f o r g e n e r a t i n g a m a g n e t i c F i e l d g r a d i e n t

w i t h i n t h e c o n t a i n e r i n w h i c h t h e m a g n e t i c f i e l d i s s t r o n g e r i n

J w w l l t h a n i n t h e -'medium most d i s t a n t ft-om t h e w a l l , , I ~ ~ +

ROBERT A. DAWSON SUPEHVtSORY PATENT EXAMINER

ART UNIT 136 ,I \ .. . --- ---.

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Tke-P_r,aperties of Magnetic Supports in Relation to.,. E Imm$iLlzed .Er_l_zy_me_,.Rf:~?.t.O.~s., Robinson et a1 . Biotechnology :and Bioengineering., V'dE;..':~X~~~li(.11.9,73)

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. OTHER DOCUMENTS (Includlng Author, Tftfe, Date, Pertinent Page, Etc.)

BioMag--Separa.to.r--.(manuf a c t u r e d by Advanced Magnet ics , I n c . , Cambridge, -MA).. - . c a t a l o g pages ( 3 . . s h e e t s )

Magnetic- . separa tor . . (rnanu.f,actured by ' Ciba-Corning Me<$caJ?y;

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. , I' . ,, .' @ - m a g n o s t i c s , Wampole, MA) - c a t a l o g cover .and-.cata.~-og..::~.~:~~

pages ( 2 s h e e t s ) ... . I

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(Form PTO-1449 [6-4])

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Magnetic separator (fma@2'%y Miltery Biotech GmbH, Gladbach,

Germany) - product information literature

@ ' Norwell, MA) - catalog pages (2 sheets)

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(Form PTO-1449 [6-4])

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OTHER DOCUMENTS (lncludlng Author, Title, Date, Pednent Pages, Etc.)

EXAMINER I DATE CONSfDEREO

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If the SMALL ENTITY is shown as YES, verify your current SMALL ENTITY status: A. If the Status is changed, pay twice the amount of the

FEE DUE shown above and notify the Patent and Trademark Office of the change in status, or

B. If the Status is the same, pay the FEE DUE shown above.

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If the SMALL ENTITY is-shown as NO: A. Pay FEE DUE shown above, or B. File verified statement of Smatl Entity Status before, or with,

payment of 1/2 the FEE DUE shown above.

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TO^, I UNITED STATES DEPARTMENT OF COMMERCE Patent and Trademark Office

Address: Box ISSUE FEE COMMISSIONER OF PATENTS AND TRADEMARKS Washington, D.C. 20231

-1 SK 1 LL"t4iWd NOTICE OF ALLOWANCE AND ISSUE FEE DUE

TIT LE OF

THE APPLICATION IDENTIFIED ABOVE HAS BEEN EXAMINED AND IS ALLOWED FOR ISSUANCE AS A PATENT. PROSECUTION ON THE MERITS IS CLOSED.

THE ISSUE FEE MUST BE PAID WITHIN THREE MONTHS FROM THE MAILING DATE OF THIS NOTICE OR THIS APPLICATION SHALL BE REGARDED AS ABANDONED. THIS STATUTORY PERIOD CANNOT BE EXTENDED.

HOW TO RESPOND TO THIS NOTICE:

IMPORTANT REMINDER: Patents issuing on applications filed on or after Dec. 12, 1980 may require payment of maintenance fees,

PTOL-E5 (REV 12-E8)(0M8 Clearance is pending) PATENT AND TRADEMARK OFFICE COPY

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. . , . . IN Th. JNITED STATES PATENT AND’T~ADEMARK OFFICE I.;

the Application of ‘ISSUE DIVISION

. Liberti BATCH: F06

1 No. 07/674,678


For: APPARATUS FOR MAGNETIC SEPARATION FEATURING EXTERNAL MAGNETIC MEANS

SUPPLEMENTAL DECLARATION

As an inventor named in the above-identified application for Letters Patent, I hereby declare that:

The subject matter described in the specification as now amended and set forth in the claims now standing allowed in said application was part of the invention described and claimed in the specification of said application and was invented before the filing of the above-identified application;

I do not know and do not believe that the same was ever known or used in the United States of America before me or our invention thereof;

I do not know and do not believe that the same was ever patented or described in any printed publication in any country before me or our invention thereof or more than one year prior to said application;

I do not know and do not believe that the same was in public use or on sale in the United States of America more than one year prior to said application;

of an inventor’s certificate issued before the date of said application in any country foreign to the United States of America on an application filed by me or my legal representatives or assigns more than twelve months prior to s a i d application; and

The invention has not been patented or made the subject

The invention has not been abandoned.

I further declare that all statements made herein of my own knowledge are true and that all statements made on information and belief are believed to be true; and further that these statements were made with the knowledge that willful false statements and the like so made are punishable by fine or imprisonment, or both, under Section 1001 of Title 18 of the United States Code and that such willful false statements may jeopardize the validity of the application or any patent issued thereon.

Date ” I 5 n P. Feeley

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ROGER W. PATRICK J. HAGAN DONALD R , PIPER. J R . V INCENT T. PACE JOSEPH M . KONIECZNY

DP COUNSEL

C. MARSHALL DANN J O H N S. .CHILD. JR. CHARLES N. O U l N N

DANN DORFMAN HERRELL AND SKILLMAN A PROFESSIONAL CORPORATION

S U I T E 720

1601 MARKET STREET

PHILADELPHIA, PA 19103-2307 - TELEPHONE

(215) 563-4100

Hon. Commissioner of Patents

Box Issue Fee Wzshington, D.C. 20231

and Trademarks

In Re the Application of ISSUE DIVISION

PAUL A. LIBERTI et al.

BATCH: F06 Serial No. 07/674,678

C O U N S E L O R S AT LAW

PATENT. TRADEMARK

A N 0 COPYRIGHT LAW

AND RELATED CAUSES - FAX

(215) 563-4044

CABLE ADORE44 SKI LPATENT

-


For: "Apparatus for Magnetic Separation Featuring External Magnetic Means

CERTIFICATE OF MAILING UNDEK 37 CFR &l.8(11)

I hereby ceriify that this correspondence is being deposited on November 3. 1992 with the United Stntes Postal Service as first class mail in an envelope properly addressed to COMMISSIONER OF PATENTS AND TRADEMARKS, Washington, D.C. 20231.

November 3. 1992 f&yJ&L&PLeq Date of Certificate Henry H. Skillman

Reg. No. 17,352

AUTHORIZATION TO CHARGE DEPOSIT ACCOUNT

In the event a fee is required and is not enclosed, or the check is improper, or the fee calculation is in error, the Commissioner is authorized to charge any underpayment or credit any overpayment to the account of the undersigned attorneys, Account No. 04-1406. A duplicate copy of this sheet is enclosed.

DANN, DORFMAN, HERRELL AND SKILLMAN, P.C.

Reg. No. 17,352. --

We are enclosing the following:

Issue Fee Transmittal PTOL-85 w/Certificate of Mailing Checks for $1170.00 and $30.00 Notification of Fee Address Supplemental Declaration

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. . . * . .3.-~Further.correspondence to be.mailed to the followingi , : . ' . . ; . ,

. . , . . . . .. Dann,, Dorfman, ,Herrell : and Skiilman ' ' , ' ., . .-

, . '*: '. ' . _ _ . . . . . .-.:

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. . 1601 Market, S t r e e t - ,Su i t e '720. ' , , , ' . : : . . . ,.

.. Philadelghta , PA 19103 . . .' . . , . . . ! , ' . '

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. . . ,4..,For.6rinting on the patent front. . Danny ;,DorfmanyL Herre: page,:list the names'of not.more.than. 3 registered patentattorneysor . . . . ? ageds,ORalternatively:.thenameo!a.' 2 ,

.firm ha{irigas:a member a"registered .;

*attorney.or agent. If no name IS

,listed: no.n,%ne.will.be printed: ,, : .. ,... 3. .

and ' Skillman

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on the patent.: Inclusion of assignee dakj Is ohly'applopriate when anassignrnent has been previously submitted to the.PTO oi Is.being submitted : this form is NOT:a subslitute for filing.an assignment:

e Is&e.fee.wiil'nor be'accepled.from anyone other than the * '

s shown by. the records of the Patent.and .Trademark Office.. . . '. a registered.at1orney or,agentj or the ,asslgneecor other party, i

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Paper Number

The Commissioner of Patents and Trademarks

Has received an application for a patent for a new and useful invention. The title and description of the invention are enclosed, The requirements of law have been complied with, and it has been determined that a patent on the invention shall be granted under the law.

Therefore, this

United States Patent Grants ro the person or persons having title to this patent the right to exclude others from making, using or selling the invention thmughout the United Stutes of America for the term of seventeen years fmm the date of this patent, subject to rhe payment of maintenance fees as provided by law.

I PTO-1584 I

Arrest

Acting Commissioner of Patents and i9ademark.v

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UNITED STATES PATENT AND TRADEMARK OFFICE

In Re U,S. Patent No. 5,186,827

Issued: February 16, 1993 *? ISSUE DIVISION Inventors: Paul A; -Libert-i et al. Serial No. 07/674,'678 February 24, 1993

Filed: March'2'5,' 1991 Q For : "Apparatus for Magnet ie

Separation Featuring External_-Magnetic Means"

missioner of Trademarks

OF CORRECTION UNDER 81.322

Certificate of Correction Form he errors made by the U.S. Patent and

Trade patent. These errors

are as follows: In the Abstract, last line, before lvmediumtl insert --test--

(see Examiner's Amendment, filed 8/3/92)

Column 3, line 20, after "Hersh et al. , I 1 insert --Clinics

(see specification, page 5, line 20). Chemica Acta, 63: 69-72 (1975). A similar--;

Co.lwn lo, line 32, after llas the@' delete glol l .

Respectfully requested,

DANN, DORFMAN, HERRELL AND SKILLMAN ,

A Professional Corporation Attorneys for Patentees

BY

Reg. No. P-36,452

Telephone: (215) 563-4100 Enc,: PTOL-1050

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IN THE UNITED STATES PATENT AND TRADEMARK OFFICE

U . S . Patent No. 5,186,827

Issued: February 16, 1993 Inventors: Paul A. Liberti et al. Serial No. 07/674,678 Filed: March 25, 1991 For: #'Apparatus for Magnetic

Separation Featuring External Magnetic Means"

To the Hon. Comndssioner of Patents and Trademarks

ISSUE DIVISION

February 2 4 , 1993

NOTICE FOR FILE

In proofreading the patent, the undersigned attorneys and/or agents respectfully request that the following corrections be noted of record in the above-identified patent.

Column 2 , lines 58-59 , ttsuperparamgnetictm should be --

Column 11, line 49, ttwold#t should be --would--; Column 12, line 1, "(now shown)tg should be --(not

Column 13, lines 67-68, lloccurrslg should be --occurs--

superparamagnetic--;

shown) --;

\

. Column 15, line 65, t*curvituregl should be --curvature--;

Column 17, line 30, tfsyrinetl should be --syringe--; Column 19, line 6, tlexpetedl* should be --expected--. This is not a request-for a Certificate of Correction.

Respectfully requested,

DANN, DORFMAN, HERRELL AND SKILLMAN A Professional Corporation Attorneys for Patentee - 1

Telephone: (215) 563-4100

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UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 5,186 827

DATED

'NVENToR(S) ' PriUl A.' Liberti; Brian P. Pe,t?ley; S)haTlesh T. Gohp.1

February 16, 1993

It is certified that m o r appears in the above-identified patent and that said Letters Patent is hereby conetted as showin below:

Q ' --- In t h e Abstract, last 1I.nc?, before "medium" inser t - - test - - - ;

Col.UmIi 3 , l ine -20, after "Hersh e t a l . ," i n s e r t --Clinicii. Chemicn Acta,

4 *>. 6 3 : . 69-72 (1975). A similar--

Column 20, l>.ne 32,--a.€ter "as the" delete "o"?.

I

PATENT NO. -7 .. . . . . . . .

No. of addl. cOpi8S . :,

MAILING ADDRESS OF SENDER:

Dam, Dorfman, Herrell and Skillman 1601 Ekrket: STreet - Suite 720 @ 30e per paw

I

. ' . Phl lade lph ia ,PA 1.91(13 +

. .

FORM P T O ' t 0 k (REV. 3-82) .

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' 0 7 APR 1993

Attorney Dann, Dorfman, Et A1 1601 Market Street, Ste. 720 Philadelphia, PA '1,9103

UNITED STATES DEPARTMENT OF COMMERCE Patent and Trademark Office ASSISTANT SECRETARY AND COMMISSIONER OF PATENTS AND TRADEMARKS Washington, D.C. 20231

I

Re: Status Regarding Certificate of Correction for U.S. Patent No. 5,186,827

Dear Attorney

On February 26, 199, we received a request for a Certificate of Correction for the above-referenced patent. We are currently experiencing a backlog of approximately 6-8 months. We appreciate your patience while we work through our backlog.

Should expedited services be required, please contact me at (703) 305-8127.

Sincerely,

Mary Allen, Manager Certificates of Correction Branch Office of Publication and Dissemination

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In re the Application of

PAUL A. LIBERTI et al.

Serial No. 07/674,678


: Group Art Unit 181

For: "Apparatus and Methods for Magnetic Separation Featuring External Magnetic : Me an s 'I

Commissioner of Patents and Trademarks Washington, DC 20231

CHANGE OF CORRESPONDENCE ADDRESS

In the matter of the above-identified patent, please address all future correspondence to:

DANN, DORFMAN, HERRELL AND SKILLMAN, P.C. 1601 Market Street, Suite 720 Philadelphia, Pennsylvania 19103-2307

Telephone: (215) 563-4100 Facsimile: (215) 563-4044


DANN, DORFMAN, HERRELL AND SKILLMAN A Professional Corporation Attorney for Applicant(s)

BY PATRICK J. HAGAN PTO Registration No. 27,643

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u N I TE D ST AT E S.-?P A RT M E NT o F c o M M E R c E /-..

Patent and Tradelllark Office

ASSISTANT SECRETARY AND COMMISSIONER OF PATENTS AND TRADEMARKS Washington, D.C. 20231

CHANGE OF ADDRESWPOWER OF ATTORNEY

F I L E L O C A T I O N 9200 S E R I A L NUMBER 07674678 P A T E N T NUMBER 5186827 - THE CORRESPONDENCE ADDRESS HAS BEEN CHANGED TO CUSTOMER # 110

THE P R A C T I T I O N E R S OF RECORD HAVE B E E N CHANGED TO CUSTOMER # 110

THE F E E ADDRESS H A S BEEN CHANGED T O CUSTOMER At 110 ~

ON 08 /31 /00 THE ADDRESS OF RECORD FOR CUSTOMER NUMBER 110 IS :

DANN DORFMAN H E R R E L L & S K I L L M A N S U I T E 720 1601 MARKET S T R E E T . P H I L A D E L P H I A PA 19103-2307

AND THE P R A C T I T I O N E R S OF RECORD FOR CUSTOMER NUMBER 110 A R E :

16703 17352 22964 27643 28833 29337 29613 31049 31812 36452 41010 43047

I..

P T O I N S T R U C T I O N S : PLEASE TAKE THE FOLLOWING A C T I O N WHEN THE CORRESPONDENCE ADDRESS H A S BEEN CHANGED TO CUSTOMER NUMBER: RECORD, ON T H E NEXT A V A I L A B L E CONTENTS L I N E OF THE F I L E J A C K E T , ’ A D D R E S S CHANGE TO CUSTOMER NUMBER’ . L I N E THROUGH THE O L D ADDRESS ON THE F I L E JACKET L A B E L AND ENTER ONLY THE ‘CUSTOMER NUMBER’ A S THE NEW ADDRESS. F I L E T H I S L E T T E R I N THE F I L E J A C K E T . WHEN ABOVE CHANGES ARE ONLY TO F E E ADDRESS AND/OR P R A C T I T I O N E R S OF RECORD, F I L E L E T T E R I N THE F I L E J A C K E T . T H I S F I L E IS A S S I G N E D TO GAU 1306. ~

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Y.S. DEPARTMENT OF COMMERCE PATENT AND T W E M A R K OFFICE

FEE RECORD SHEET

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Documents

:i~~~RI~~~~rjmorris/sciev.06/SIMULATIONS/stanlaw_5... · 1 5,186,827 2 says, involve the use of labeled antibodies instead of labelled analyte. In performing an immunometrk assay,

:i~RI~~rjmorris/sciev.06/SIMULATIONS/stanlaw_5... · 1 5,186,827 2 says, involve the use of labeled antibodies instead of labelled analyte. In performing an immunometrk assay,